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EP4004018A1 - Modifizierte humane cytomegalovirus-proteine - Google Patents

Modifizierte humane cytomegalovirus-proteine

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Publication number
EP4004018A1
EP4004018A1 EP20747135.0A EP20747135A EP4004018A1 EP 4004018 A1 EP4004018 A1 EP 4004018A1 EP 20747135 A EP20747135 A EP 20747135A EP 4004018 A1 EP4004018 A1 EP 4004018A1
Authority
EP
European Patent Office
Prior art keywords
hcmv
protein
modified
seq
gcn4
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20747135.0A
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English (en)
French (fr)
Inventor
Marco BIANCUCCI
Sumana CHANDRAMOULI
Newton Muchugu WAHOME
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
GlaxoSmithKline Biologicals SA
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Publication date
Application filed by GlaxoSmithKline Biologicals SA filed Critical GlaxoSmithKline Biologicals SA
Publication of EP4004018A1 publication Critical patent/EP4004018A1/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to recombinant human cytomegalovirus (HCMV) gB proteins.
  • HCMV Human cytomegalovirus
  • At least twenty viral proteins are expressed on the HCMV envelope, including glycoproteins B, L and O (gB, gL and gO, respectively), and proteins UL128, UL130 and UL131A.
  • glycoproteins B, L and O gB, gL and gO, respectively
  • proteins UL128, UL130 and UL131A The glycoprotein gB trimer and gH/gL heterodimer form a core fusion machinery.
  • HCMV cell entry consists of a two-step mechanism: receptor binding and membrane fusion. While the gH/gL/gO and gH/gL/UL128/UL130/UL131A (corresponding to the HCMV pentamer) oligomers recognize their cognate receptors on cell membranes and regulate cell tropism, the gB trimer mediates membrane fusion and acts as a viral fusogen.
  • viral fusogens are divided into three classes, with HCMV gB belonging to class III.
  • Viral fusogens are anchored into the viral envelope and reside in a metastable pre-fusion state on the viral membrane.
  • viral fusogens undergo a conformational rearrangement, switching to a stable post-fusion conformation and facilitating the merging of the viral lipid envelope with the host cell membrane.
  • the present inventors designed constructs that produce HCMV gB proteins in non-post- fusion conformation, or that interfere with the transition of the HCMV gB protein from pre-fusion to post-fusion conformation. Without wishing to be bound by theory, it is believed that the“non-post- fusion” conformation referred to herein is the“pre-fusion” conformation. To do so, the inventors replaced the amino acid residues c-terminal to the residue corresponding to K724 of SEQ ID NO: 1 with a heterologous trimerization domain that promotes the formation of stable trimers, such as a GCN4 Leucine Zipper domain.
  • the gB protein was truncated by deleting the amino acids c-terminal to the residue corresponding to K724 of SEQ ID NO: 1 , thus removing a part of the membrane proximal region as well as both the transmembrane domain and the cytoplasmic domain.
  • a heterologous multimerization domain such as GCN4 Leucine Zipper domain (GCN4 domain) was added to the C-terminus of the truncated gB protein. This modification is sufficient to produce gB proteins in non-post-fusion conformation.
  • the present result is surprising because GCN4 domain insertions into other herpesvirus gB proteins (those from HSV and EBV) have been reported as failing to either stabilize the recombinant gB protein in a non-post-fusion conformation orto interfere with the transition from pre-fusion to post-fusion conformation. See Vitu et al. 2013 J. Mol. Biol. 425(11): 2056-2071 (regarding HSV gB) and Backovic et al. 2007 Virology 368(1): 102- 1 13 (regarding EBV gB). The present result is particularly surprising in view of at least Vitu et al. (2013 J. Mol. Biol. 425(11): 2056-2071) who postulate that the transmembrane region and cytodomain are required for the stability of prefusion conformation.
  • the present inventors further modified the gB molecule with one or more proline substitution(s) (such as one or two proline substitutions) (in particular, a proline substitution at a residue corresponding to any one of N478-R511 ; or more specifically A503-R511 ) (see Table 2 and the Examples herein) which substitutions increased the percentage of molecules produced in the non-post-fusion conformation.
  • proline substitutions such as one or two proline substitutions
  • substitutions increased the percentage of molecules produced in the non-post-fusion conformation.
  • the gB molecule may additionally be modified with any helix-breaking substitution(s) or insertion(s) of one or more G, S, A, or P residues.
  • Such helix-breaking modifications occur at one or more (e.g., one or two) residues corresponding to N478-R511 ; or more specifically A503-R511 (numbered according to SEQ ID NO:1). See Table 2 herein.
  • c-terminal truncation and addition of a heterologous trimerization domain, as described herein, in an HCMV gB protein is sufficient to disrupt the transition of the viral gB protein from pre-fusion to post-fusion conformation or to stabilize the gB protein in a non-post-fusion conformation.
  • the heterologous trimerization domain is located c-terminally adjacent to the residue corresponding to K724 of SEQ ID NO: 1 .
  • the present inventors further describe incorporating an additional modification (a proline substitution) of at least one of the following (such as one or two of the following), to increase production of HCMV gB proteins in non-post-fusion conformation:
  • Protein expression of the modified HCMV gB proteins as described herein increases the percentage of gB proteins produced in the non-post-fusion conformation (as compared to production of non-modified HCMV gB proteins).
  • One aspect of the present invention provides a modified HCMV gB protein comprising a heterologous trimerization domain operably linked c-terminal to the residue corresponding to 724 numbered according to SEQ ID NO: 1 (specifically K724 numbered according to SEQ ID NO: 1).
  • the modified gB protein herein may be truncated, i.e. , not comprise the native amino acid residues c-terminal to the residue corresponding to 724, numbered according to SEQ ID NO: 1 (specifically, does not comprise the native amino acid residues corresponding to 725-907 of SEQ ID NO: 1).
  • Such native amino acid residues form a portion of the MPR domain, the transmembrane domain, and the cytoplasmic domain (FIG. 1).
  • the modified HCMV gB protein has an amino acid linker sequence between the residue corresponding to 724, numbered according to SEQ ID NO: 1 (i.e., the c- terminal-most residue of the truncated MPR) and the GCN4 domain.
  • Suitable linker sequences include a single amino acid (such as a single proline, serine, glycine or alanine residue), two amino acid residues (such as two proline (P), serine (S), glycine (G) or alanine (A) residues, or any combination thereof), or any short amino acid sequence that functions as a linker sequence.
  • the heterologous trimerization domain is a GCN4 Leucine Zipper domain (GCN4); a suitable GCN4 domain comprises or consists of the amino acid sequence provided as SEQ ID NO: 2.
  • GCN4 Leucine Zipper domain GCN4
  • suitable GCN4 domain comprises or consists of the amino acid sequence provided as SEQ ID NO: 2.
  • Additional multimerization domains that drive the symmetric self-assembly of trimers of soluble proteins are known in the art (Engel et al. 2000 Matrix Biology 19.4:283-288) , and may be used in the present invention.
  • suitable multimerization domains include: (1) the GCN4 leucine zipper (Harbury et al. 1993 Science 262: 1401-1407); (2) the phage T4 fibritin foldon (Miroshnikov et al.
  • the modified HCMV gB protein comprises a substitution modification of one or more furin cleavage site amino acid, including but not limited to R457S and R460S substitutions (numbered per SEQ ID NO:1).
  • the modified HCMV gB protein comprises a substitution modification of one or more fusion loop amino acids, including but not limited to substitution modifications at FL1 residues155-157 and/or FL2 residues 240-242 (numbered per SEQ ID NO: 1 ), such as Y155G, I 156H, H157R, W240F, L241 F, and Y242H (numbered per SEQ ID NO: 1).
  • the modified FICMV gB protein comprises a purification tag (e.g., a 6- histidine purification tag) and may also comprise a cleavage linker (e.g., a TEV cleavage linker).
  • a purification tag e.g., a 6- histidine purification tag
  • a cleavage linker e.g., a TEV cleavage linker
  • the modified FICMV gB protein comprises a proline substitution of one or more Domain II I Coiled-coil (Dl llcc) Region amino acid residues corresponding to N478-R51 1 numbered according to SEQ ID NO: 1 .
  • Dll lcc amino acid residues are any one or more (such as one or two) of the residues listed within Table 2.
  • the modified FICMV gB protein comprises a helix-breaking substitution (amino acid substitution of one or more G, S, A, or P residues) for one or more Domain I II Coiled-coil (Dll lcc) Region amino acid residues corresponding to N478-R51 1 numbered according to SEQ ID NO: 1 .
  • Dl llcc amino acid residues are any one or more (such as one or two) of the residues listed within Table 2.
  • the modified FICMV gB protein is operably linked to a carrier.
  • the carrier is a nanoparticle.
  • the nanoparticle is, or is derived from, lumazine synthase or ferritin proteins, or other bacterial, viral proteins, or cte novo designed symmetric carriers (Yeates et al. 2017 Annual Reviews of Biophysics 46:23-42) that form nanoparticles.
  • isolated nucleic acids comprising a polynucleotide sequence encoding the modified FICMV gB proteins herein.
  • the nucleic acid may be an RNA.
  • the RNA may be self-replicating RNA, optionally an alphavirus replicon.
  • An alphavirus replication particle (VRP) may comprise the alphavirus replicon.
  • immunogenic compositions comprising a modified FICMV gB protein, nucleic acid , or VRP of herein.
  • Immunogenic compositions may comprise an adjuvant, and the adjuvant may comprise aluminum salt, a TLR7 agonist, TLR4 agonist, MPL, 3D-MPL, saponin , or an oil-in-water emulsion.
  • Immunogenic compositions herein may comprise a modified FICMV gB protein and at least one additional protein or nucleic acid antigen .
  • the at least one additional antigen may be an FICMV protein, or immunogenic fragment thereof, such as gO, gH, gL, pUL128, pUL130, pUL131 , pp65, IE1 , or a combination thereof.
  • a recombinant vector comprising a nucleic acid described herein.
  • an isolated host cell comprising a nucleic acid as described herein .
  • the nucleic acid may comprise a polynucleotide that is DNA and such DNA may be stably incorporated into the genomic DNA of the host cell.
  • the host cell may be a mammalian cell.
  • the mammalian cell may be a CHO cell or HEK-293 cell.
  • a cell culture comprising a host cell as described herein.
  • processes of producing a modified HCMV gB protein comprising culturing a host cell undersuitable conditions, and optionally then collecting the modified gB protein from the cultured host cell(s), and optionally purifying the modified gB protein.
  • processes of producing a population of host cells comprising culturing host cells under suitable conditions for expression of a modified HCMV gB protein as described herein and optionally purifying the modified gB protein, wherein at least 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, or 45% of the purified modified gB proteins are in pre- fusogenic conformation.
  • a population of host cells wherein at least 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, or 45% of the modified HCMV gB proteins expressed from said host cells are in pre-fusogenic conformation.
  • a pharmaceutical composition may comprise such antibody, or antigen-binding fragment thereof.
  • Another aspect provides uses of the modified HCMV gB proteins, nucleic acids, VRPs, immunogenic compositions, vectors, antibodies or antibody fragments, or pharmaceutical compositions described herein for the prevention or treatment of HCMV infection, for inducing an immune response against HCMV, for the manufacture of a medicament for inducing an immune response against HCMV, or for the manufacture of a medicament for inhibiting HCMV entry into a cell.
  • kits for identifying the presence of a modified HCMV gB protein within a composition for the purification of a modified HCMV gB protein from a composition, for detecting a modified HCMV gB protein in pre-fusion conformation or a non-post-fusion conformation, or for the prevention or treatment of HCMV virus infection; comprising the antibody or antigen-binding fragment as described herein.
  • kits may include instructions for using the antibody or antigen-binding fragment thereof.
  • FIG. 1 is a schematic representation of the HCMV gB protein from the Merlin strain. Abbreviations therein are as follows: “TM” meaning transmembrane domain; “Cyto” meaning cytoplasmic domain;“SP” meaning signal peptide;“MPR” meaning membrane proximal region;“I” meaning Domain I;“II” meaning Domain II;“III” meaning Domain III;“IV” meaning Domain IV; and “V” meaning Domain V.
  • TM transmembrane domain
  • Cyto meaning cytoplasmic domain
  • SP meaning signal peptide
  • MPR membrane proximal region
  • I meaning Domain I
  • III meaning Domain II
  • III meaning Domain III
  • IV meaning Domain IV
  • V meaning Domain V.
  • the amino acid numbering and residues are based on HCMV Merlin strain gB sequence SEQ ID NO: 1 ( see also Chandramouli et al. , 2015 Nat. Commun.
  • Circled‘G’ represents N glycosylation site. Fusion loops, Furine Cleavage Site, and Dill Coiled-coil region are indicated according to the HCMV Merlin strain gB sequence (SEQ ID NO:1).
  • FIG. 2A and FIG. 2B provide an alignment spanning the Domain III Coiled-coil (Dlllcc) Region and Membrane Proximal Region (MPR) amino acid residues of (top to bottom) HCMV Merlin strain gB (SEQ ID NO: 1), HCMV AD169 strain gB (SEQ ID NO: 6). Residue numbering is provided with respect to SEQ ID NO: 1 and, in this way, demonstrates what is meant by an HCMV AD169 gB amino acid residue that“corresponds to” a residue“numbered according to SEQ ID NO: 1”.
  • Dlllcc Domain III Coiled-coil
  • MPR Membrane Proximal Region
  • FIG. 3A depicts the results of NanoDSF analysis (raw data and first derivative, Ratio 350 nm/330 nm) conducted on gB’-GCN4 and two gB’ proteins (denoted gB’1 and gB’2 therein, which are based on HCMV AD169 strain and Merlin strain, respectively).
  • gB’GCN4 provides two peaks (one at a melting temperature of about 66.3 degrees Celsius and the other at a melting temperature of about 78.9 degrees Celsius) whereas gB’1 only provides one peak (at 77.2 degrees Celsius) and gB’2 only provides one peak (at 78.9 degrees Celsius).
  • the labels on the left side are, from top to bottom, 0.85, 0.80, 0.75, and 0.70, and represent the ratio of the fluorescence signal at 350 nm/330 nm.
  • the temperature (degrees Celsius) labels along the bottom are, from left to right, 30, 40, 50, 60, 70, 80, and 90.
  • Lines corresponding to gB’-GCN4 are indicated by a solid triangle; those corresponding to gB’1 by a solid circle; those corresponding to gB’2 by an open circle.
  • FIG. 3B depicts NanoDSF results for gB’-GCN4 only and confirms that gB’-GCN4 provides two peaks: peak I having a melting temperature of 78.9 degrees Celsius and another, peak II (left- hand peak), having a melting temperature at 66.3 degrees Celsius (12.6 degrees below that of peak I).
  • the labels on the left side are, from top to bottom, 0.95, 0.90, 0.85, 0.80, and 0.75, and represent the ratio of the fluorescence signal at 350 nm/330 nm.
  • the temperature (degrees Celsius) labels along the bottom are, from left to right, 30, 40, 50, 60, 70, 80, and 90.
  • FIG. 4A summarizes, for differently modified gB proteins, what percentage of the modified gB protein had a melting temperature as at peak I and what percentage had a melting temperature as at peak II, determined via the results of SEC-HPLC and NanoDSF analysis.“WT” indicates gB’- GCN4 (SEQ ID NO:3); Q510P, R511 P, R512P, T513P, L514P, E515P, V516P, and F517P indicate gB’-GCN4 (SEQ ID NO:3) with the indicated proline substitution (numbered according to SEQ ID NO:1).
  • FIG. 4B summarizes the ratio of modified gB proteins having a melting temperature as at peak II to those having a melting temperature as at peak I.“WT” indicates gB’-GCN4 (SEQ ID NO:3); additional proteins indicated as in FIG. 4A.
  • FIG. 5 depicts HCMV Merlin strain gB as a trimer and indicates alpha helices residues D509 (three shown due to trimer), Q510, R511 , R512, T513, L514, E515, V516, and F517 (numbered according to SEQ ID NO: 1). Also shown is the magnesium ion (Mg2+) between the alpha helices. Residue Q510 is denoted with an arrow.
  • FIG. 6A depicts quantification (in percent) of Peak I and Peak II population by SEC-HPLC of gB’-A505P-GCN4, gB’-W506P-GCN4, gB’-C507P-GCN4, gB’-D509P-GCN4, and gB’-D509P- Q510P-GCN4 molecules.
  • Dotted lines indicate the Peak II percentage of gB’-GCN4 (designated as “WT” therein) and of the gB’-GCN4-Q510P mutant that is described elsewhere herein (designated as“Q510P” therein).
  • FIG. 6B depicts Peak ll/Peak I ratio gB’-A505P-GCN4, gB’-W506P-GCN4, gB’-C507P- GCN4, gB’-D509P-GCN4, and gB’-D509P-Q510P-GCN4 molecules. Dotted lines indicate the Peak ll/Peak I ratio of gB’-GCN4 (designated as“WT” therein) and of the gB’-GCN4-Q510P mutant that is described elsewhere herein (designated as“Q510P” therein).
  • FIG. 7A depicts the elution profiles under SEC-HPLC analysis ofgB698-GCN4 (left/lower line with only one peak) and EBV-gB-GCN4 (right/top line with two peaks) molecules.
  • FIG. 7B depicts the thermal transition under NanoDSF analysis of gB698-GCN4 and EBV- gB-GCN4 molecules.
  • FIG. 8 depicts the thermal transition of Peak I and Peak II of gB’-GCN4-Q510P and LVL759 (control) under NanoDSC.
  • FIG. 9 provides a schematic representation of what is believed to be the transition from pre- to postfusion HCMV gB (schematic adapted from Nishimura & Mori 2019 Ad. Vir. Res. 104: 283- 312).
  • the box on the Globular State gB shows where the GCN4 was placed and the arrow on the Globular State shows the location of the Q510P mutation described herein.
  • FIG. 10A depicts the quantification of Peak II to Peak I ratio observed for gB’-GCN4 (labeled as“WT”) and molecules (1)-(8) from Example 1 following SEC-HPLC analysis (see Examples 1 and 5).
  • FIG. 10B depicts the quantification of Peak II to Peak I ratio observed for gB’-GCN4 (labeled as“WT”) and molecules (9)-(25) following SEC-HPLC analysis (see Example 5).
  • Top dotted line indicates the Peak IhPeak I ratio observed for the gB’-Q510P-GCN4 molecule.
  • Bottom dotted line indicates the Peak IhPeak I ratio observed for the gB’-GCN4 molecule.
  • FIG. 10C depicts the quantification of Peak II to Peak I ratio observed for gB698-GCN4 (labeled as“WT AD169”) and molecules (26)-(27) following SEC-HPLC analysis (see Example 5).
  • Top dotted line indicates the Peak IhPeak I ratio observed for the gB’-Q510P-GCN4 molecule.
  • Bottom dotted line indicates the Peak IhPeak I ratio observed for the gB’- GCN4 molecule.
  • This supposed pre-fusion gB model has a Christmas tree-like conformation, where the apical part seems to belong to the coiled-coil region of Domain III (which is usually buried in the post-fusion conformation) (FIG. 1).
  • the low-resolution model by Si Z. et al. does not allow for structural design of an HCMV gB molecule (e.g., one that is not in post-fusion conformation) and, in particular, structural design of a stable HCMV gB molecule in the pre-fusion conformation.
  • the inventors have discovered that certain modifications introduced to HCMV gB protein stabilize it in a non-post-fusion conformation, or interfere with the protein’s transition from pre-fusion to post-fusion conformation.
  • the gB protein is truncated within the Membrane Proximal Region (MPR) and operably linked to a heterologous trimerization domain (such as a GCN4 Leucine Zipper domain), optionally with an amino acid linker sequence (such as two proline residued) inserted between the truncated MPR and the heterologous trimerization domain.
  • MPR truncation comprises a deletion of the amino acids that are c-terminal to the residues listed in Table 1.
  • the modified HCMV gB molecules may be further modified in the Domain III Coiled-coil Region, to include any helix-breaking amino acid substitution(s) or insertion(s), such as with one or more G, S, A, or P residues.
  • substitution of one or more of the residues listed in Table 2 to proline is one such modification.
  • HCMV gB is an envelope glycoprotein B having numerous roles, one of which is the involvement in the fusion of the cytomegalovirus with host cells. It is encoded by the UL55 gene of HCMV genome.
  • the size of the native form of HCMV gB depends on the size of the open reading frame (ORF) which may vary a little according to the strain. For example, the ORF of AD169 strain, which is 2717 bp long, encodes a full length gB of 906 amino acids, whereas the ORF of Towne and Merlin strains encode a full length gB of 907 amino acids.
  • the present invention is applicable to gB proteins originating from any HCMV strain, in order to facilitate its understanding, when referring to amino acid positions in the present specification, the numbering is given in relation to the amino acid sequence of the gB protein of SEQ ID NO:1 originating from the clinical isolate Merlin strain, unless otherwise stated.
  • the present invention is not, however, limited to the HCMV Merlin strain.
  • the modified HCMV gB protein comprises a helix-breaking substitution (amino acid substitution of one or more G, S, A, or P residues) for one or more Domain III Coiled-coil (Dlllcc) Region amino acid residues corresponding to N478-R511 (numbered according to SEQ ID NO: 1).
  • Dllcc amino acid residues include any one or more (such as one or two) of the residues listed within Table 2.
  • Suitable proline substitutions include K723P of HCMV AD169 strain gB amino acid sequence SEQ ID NO: 6 (corresponding to K724 residue of SEQ ID NO:1).
  • Amino acid residues corresponding to position 478 of SEQ ID NO: 1 from SEQ ID NO: 6 are set forth below in Table 2, numbered according to (or“with respect to’’) SEQ ID NO: 1 .
  • a gB residue that“corresponds to’’ a residue in a different HCMV gB may have a different position number, may be a different amino acid, or both (see also FIGs. 2A-2B).
  • the HCMV AD- 169 strain gB Fusion Loop 1 FL1 sequence 155 YIY 157 “corresponds to’’ or is“comparable to’’ the FICMV Merlin strain gB FL1 sequence 155 YIH 157 (Y 157 from AD-169 strain gB corresponding to H 157 of Merlin strain gB) (compare WO 2012/049317 (corresponding to US 2013/0216613) and WO
  • Orientation within a polypeptide is generally recited in an N-terminal to C-terminal direction, defined by the orientation of the amino and carboxy moieties of individual amino acids. Polypeptides are translated from the N-terminal or amino-terminus towards the C-terminal or carboxy-terminus.
  • the native form of FICMV Merlin strain gB is set forth in FIG. 1 and generally contains, in the
  • amino acid signal sequence or signal peptide here designated as residues 1-22 of SEQ ID NO: 1 , but designated by some as residues 1-24 of SEQ ID NO: 1 ( see UniProtKB Accession No.
  • F5FIB53 F5FIB53
  • the HCMV gB protein forms a monomeric trimer (comprising three gB proteins, also referred to as subunits) that can be used as an antigen against HCMV.
  • the monomeric trimer comprises an exposed hydrophobic surface, which can cause significant problems in both antigen production and purification.
  • the hydrophobic surface can cause aggregation of recombinantly produced gB protein (e.g., two monomeric trimers can form a dimeric trimer via the hydrophobic surface, which may cause production problems).
  • the hydrophobic surface also causes the monomeric gB trimer to adhere to the host cell (e.g., to cell membrane, ER membrane, other hydrophobic proteins, aggregated proteins, etc.).
  • one or more residues within either or both of Fusion Loop 1 and Fusion Loop 2 corresponding to residues 155-157 and 240-242, respectively, of SEQ ID NO: 1 may be modified (e.g., by amino acid residue substitution) (see Chandramouli et al. 2015 Nat. Comm. 6(8176): 1-12; WO 2012/049317 (corresponding to US 2013/0216613) and WO 2016/092460 (corresponding to US 2018/0265551).
  • Proteins (or polypeptides) herein may be modified to carry further modifications, such as for instance, modifications at endoproteolytic cleavage sites so that said sites are made ineffectual.
  • modifications at endoproteolytic cleavage sites so that said sites are made ineffectual.
  • the furin cleavage site located around amino acids 457 to 460 of the sequence set forth in SEQ ID NO:1 , or at a corresponding position in other gB proteins originating from different HCMV strains may be modified. See WO 2012/049317 (corresponding to US 2013/0216613) and WO 2016/092460 (corresponding to US 2018/0265551).
  • fragment in reference to a polypeptide, refers to a portion (that is, a subsequence) of a polypeptide.
  • An“immunogenic fragment” of a gB protein refers to a fragment that retains at least one immunogenic epitope (e.g., a predominant immunogenic epitope or a neutralizing epitope) of the full-length gB protein.
  • immunogenic epitope e.g., a predominant immunogenic epitope or a neutralizing epitope
  • AD antigenic domains
  • the immunogenic fragment described herein comprises an antigenic domain selected from the group consisting of AD-1 , AD-2, AD-3, AD-4, AD-5, and a combination thereof.
  • An “immunogenic fragment” of a modified gB protein still comprises all of the modifications made to, or present in, the corresponding portion of the full length, referenced modified gB protein.
  • the modified gB proteins of the present invention maintain immunogenic properties or epitope(s), so it is a further object of the present invention to utilize the modified polypeptides and modified fragments thereof in polypeptide/antibody interactions.
  • the invention therefore provides antibodies which recognise a modified HCMV gB protein as described herein and, further, an antibody that is specific for such modified HCMV gB protein.
  • the antibodies of the present invention may be a monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibodies), labelled antibody, or antibody fragment so long as they exhibit the desired antigen-binding activity.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SFI, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • the modified FICMV gB proteins herein may be used to identify antibodies using in vitro selection methods, such as phage display using diverse antibody libraries.
  • the invention also provides a method for raising antibodies using a modified gB protein of the invention.
  • An antibody or antibody fragment of the invention may be human or humanised.
  • the antibodies of the invention may be used in a diagnostic assay and may be labelled directly or indirectly.
  • the antibodies of the invention may be used in therapy, for example in the treatment FICMV infection and may be in the form of neutralizing antibodies, which can inhibit or neutralize a gB protein’s biological activity.
  • glycan attachment of a glycan to a gB protein creates a physical barrier (as well as a more hydrophilic surface) to reduce aggregation/adhesion via the hydrophobic surface.
  • Glycosylation sites can be introduced into desired locations by suitable modification of amino acid sequences of the gB protein. See WO 2016/092460 (corresponding to US 2018/0265551).
  • N-linked glycosylation sites comprising the N-X-S/T/C motif, are introduced.
  • the motif is N-X- S T.
  • X is not proline.
  • sites for O-linked glycosylation can also be added.
  • O-linked glycosylation the carbohydrate moiety is linked to the hydroxyl oxygen of serine and threonine.
  • O-linked glycosylation also occurs at tyrosine, 5-hydroxylysine, and 4- hydroxyproline.
  • Amino acids may be substituted for (replaced by) a‘‘hydrophilic’’ or‘‘comparatively more hydrophilic’’ amino acid.
  • the hydrophobicity of a particular amino acid sequence can be determined using a hydrophobicity scale, such as the Kyte and Dolittle scale (Kyte et al. 1982. J. Mol. Bio. 157: 105-132). Flydrophobicity of an amino acid sequence or a fragment thereof is dictated by the type of amino acids composing this sequence or a fragment thereof.
  • Amino acids are commonly classified into distinct groups according to their side chains. For example, some side chains are considered non-polar, i.e. hydrophobic, while some others are considered polar, i.e. hydrophilic.
  • Alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F) and tryptophan (W) are considered to be hydrophobic amino acids, while serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y), cysteine (C), lysine (K), arginine (R), histidine (FI), aspartic acid (D) and glutamic acid (E) are considered to be polar amino acids. Regardless of their hydrophobicity, amino acids are also classified into subgroups based on common properties shared by their side chains.
  • phenylalanine, tryptophan and tyrosine are jointly classified as aromatic amino acids and will be considered as aromatic amino acids within the meaning of the present invention .
  • Aspartate (D) and glutamate (E) are among the acidic or negatively charged amino acids, while lysine (K), arginine (R) and histidine (H) are among the the basic or positively charged amino acids, and they will be considered as such in the sense of the present invention.
  • Hydrophobicity scales are available which utilize the hydrophobic and hydrophilic properties of each of the 20 amino acids and allocate a hydrophobic score to each amino acid, creating thus a hydrophobicity ranking.
  • the Kyte and Dolittle scale may be used (Kyte et al. 1982. J. Mol. Bio. 157: 105-132). This scale allows one skilled in the art to calculate the average hydrophobicity within a segment of predetermined length . Accordingly, hydrophobic regions in an amino acid sequence may be identified by the skilled person as potential targets for modification in accordance with the present invention. The ability of the modification of said regions to induce an improved product profile of the resulting modified protein, i.e. favoring the monomeric trimers proportion within the population, may then be tested as described below.
  • the modification of a hydrophobic region may be an addition, deletion, or substitution of amino acid(s) within the hydrophobic surface (e.g., substituting hydrophobic amino acids with polar amino acids).
  • a modification of HCMV gB protein residue C246 of SEQ ID NO: 1 may be introduced .
  • Such modification can be, e.g ., C246S, C246A, or C246G (or corresponding residues).
  • C246 is an unpaired cysteine and modifying this unpaired cysteine can reduce the undesired formation of inter-molecular disulfide bonds.
  • the HCMV gB protein may include a signal peptide at the N-terminus.
  • a signal peptide can be selected from among numerous signal peptides known in the art, and is typically chosen to facilitate production and processing in a system selected for recombinant expression ofthe HCMV gB protein (or immunogenic fragment thereof).
  • signal peptides are 5-30 amino acids long, and are typically present at the N-terminus of a newly synthesized protein.
  • the core of the signal peptide generally contains a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptides begin with a short hydrophilic (usually positively charged) stretch of amino acids, which may help to enforce proper topology of the polypeptide during translocation.
  • hydrophilic amino acids usually positively charged
  • At the end of the signal peptide (C-terminus) there is typically a stretch of hydrophilic amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the signal peptide included in the modified HCMV gB peptide is the signal peptide naturally present in the native HCMV gB protein, where“native” means naturally occurring or as found in nature.
  • the signal peptide is located at residues 1-22 (i.e., 1-22 of SEQ ID NO:1).
  • Signal peptide from other HCMV strains can be identified by sequence alignment.
  • the signal peptide may be a heterologous sequence in that the sequence arises from a protein distinct from gB.
  • Exemplary signal peptides suitable for use in the context ofthe HCMV gB protein (or an immunogenic fragment thereof) described herein include signal peptides of tissue plasminogen activator (tPA), Herpes Simplex Virus (HSV) gD protein, human endostatin, HIV gp120, CD33, human Her2Neu, gp67, or Epstein Barr Virus (EBV) gp350.
  • the signal peptide can be non-native and may comprise modifications, such as substitutions, insertions, or deletions of amino acids. In particular, modifications can be introduced at C-terminal part of the signal peptide.
  • the modified HCMV gB proteins (or immunogenic fragment thereof) of the invention can include the addition of an amino acid sequence that constitutes a tag, which can facilitate detection (e.g. an epitope tag for detection by monoclonal antibodies) and/or purification (e.g. a polyhistidine-tag to allow purification on a nickel-chelating resin) of the proteins.
  • a tag which can facilitate detection (e.g. an epitope tag for detection by monoclonal antibodies) and/or purification (e.g. a polyhistidine-tag to allow purification on a nickel-chelating resin) of the proteins.
  • affinity-purification tags include, e.g., 6xHis tag (hexahistidine, binds to metal ion), maltosebinding protein (MBP) (binds to amylose), glutathione-S-transferase (GST) (binds to glutathione), FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 7), binds to an anti-flag antibody), Strep tag (Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 8), or Trp-Ser-His-Pro-GIn-Phe-Glu- Lys (SEQ ID NO: 9), or Trp-Ser-His-Pro-GIn-Phe-Glu-Lys-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-G
  • cleavable linkers may be used. This allows for the tag to be separated from the purified complex, for example by the addition of an agent capable of cleaving the linker.
  • an agent capable of cleaving the linker A number of different cleavable linkers are known to those of skill in the art. Such linkers may be cleaved forexample, by irradiation ofa photolabile bond or acid-catalyzed hydrolysis.
  • polypeptide linkers which incorporate a protease recognition site and which can be cleaved by the addition of a suitable protease enzyme including, for example, a Tobacco Etch Virus (TEV) cleavage linker (or cleavage site).
  • TEV Tobacco Etch Virus
  • the nucleic acid can express a modified HCMV gB protein (or an immunogenic fragment thereof) as described herein.
  • the modified HCMV gB protein may then be secreted from the host cell.
  • Suitable host cells include, for example, insect cells (e.g., Aedes aegypti, Autographs californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia nf), mammalian cells (e.g., human, nonhuman primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster)), avian cells (e.g., chicken, duck, and geese), bacteria (e.g., E.
  • insect cells e.g., Aedes aegypti, Autographs californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia nf
  • mammalian cells e.g., human, nonhuman primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster)
  • yeast cells e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica
  • Tetrahymena cells e.g., Tetrahymena thermophila
  • the host cell should be one that has enzymes that mediate glycosylation.
  • Bacterial hosts are generally not suitable for such modified proteins, unless the host cell is modified to introduce glycosylation enzymes; instead, a eukaryotic host, such as insect cell, avian cell, or mammalian cell should be used.
  • Suitable insect cell expression systems such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA.
  • a suitable baculovirus expression vector such as pFastBac (Invitrogen) is used to produce recombinant baculovirus particles.
  • the baculovirus particles are amplified and used to infect insect cells to express recombinant protein.
  • Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)).
  • Avian cell expression systems are also known to those of skill in the art and described in, e.g., U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent No. EP 0787180B; European Patent Application No. EP03291813.8; WO 03/043415; and WO 03/076601.
  • Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx cells), chicken embryonic fibroblasts, chicken embryonic germ cells, duck cells (e.g., AGE1 .CR and AGE1.CR.plX cell lines (ProBioGen) which are described, for example, in Vaccine 27:4975-4982 (2009) and W02005/042728), EB66 cells, and the like.
  • chicken embryonic stem cells e.g., EBx cells
  • chicken embryonic fibroblasts e.g., chicken embryonic germ cells
  • duck cells e.g., AGE1 .CR and AGE1.CR.plX cell lines (ProBioGen) which are described, for example, in Vaccine 27:4975-4982 (2009) and W02005/042728
  • EB66 cells e.g., EB66 cells, and the like.
  • the host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the inserted polynucleotide sequences.
  • the culture conditions such as temperature, pH and the like, are typically those previously used with the host cell selected for expression and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein.
  • Expression products corresponding to the nucleic acids of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like.
  • vectors which direct high level expression of proteins that are readily purified are favorably employed.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the coding sequence of interest, e.g., a polynucleotide of the invention as described above, can be ligated into the vector in-frame with sequences forthe amino-terminal translation initiating methionine and the subsequent seven residues of beta-galactosidase producing a catalytically active beta galactosidase fusion protein; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison Wl), in which the amino-terminal methionine is ligated in frame with a histidine tag; and the like.
  • BLUESCRIPT Multifunctional E. coli cloning and expression vectors
  • yeast such as Saccharomyces cerevisiae
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used for production of the desired expression products.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH
  • PGH protein oxidase
  • a host cell is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion, including, but not limited to, glycosylation, acetylation, carboxylation, phosphorylation, lipidation, and acylation.
  • Post- translational processing for example, which cleaves a precursor form into a mature form of the protein (for example, by a furin protease) is optionally performed in the context of the host cell.
  • Different host cells such as 3T3, COS, CHO, HeLa, HEK, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • modified gB protein encoded by the nucleic acids disclosed herein stable expression systems are typically used.
  • cell lines which stably express a modified HCMV gB protein of the invention are obtained by introducing into the host cell expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells are allowed to grow for 1 -2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • resistant groups or colonies of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Host cells transformed with a nucleic acid encoding a modified HCMV gB protein are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • the secreted polypeptide product is then recovered from the culture medium and purified.
  • cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Eukaryotic or microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption.
  • Modified gB proteins can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any ofthe tagging systems noted herein), hydroxyapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps.
  • HPLC high performance liquid chromatography
  • the host cells are mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster).
  • mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK-293 cells,, NIH-3T3 cells, 293-T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL-160), Madin-Darby bovine kidney (“MDBK”) cells, Madin-Darby canine kidney (“MDCK”) cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), baby hamster kidney (BHK) cells, such as BHK21-F
  • BHK baby
  • the host cell is a CHO cell.
  • the nucleic acid encoding the modified HCMV gB protein (or immunogenic fragment thereof) described herein is stably integrated into the genome of the CHO cell.
  • CHO cell lines are also available from European Collection of Cell Cultures (ECACC), or American Type Culture Collection (ATCC), such as CHO cell lines hCBE11 (ATCC PTA-3357TM), E77.4 (ATCC PTA-3765TM), hLT-B: R-hG1 CHO #14 (ATCC CRL-11965TM), MOR- CHO- MORAb-003-RCB (ATCC PTA-7552TM), AQ.C2 clone 11 B (ATCC PTA-3274TM), AQ.C2 clone 11 B (ATCC PTA-3274TM), hsAQC2 in CHO-DG44 (ATCC PTA-3356TM), xrs5 (ATCC CRL- 2348TM), CHO-K1 (ATCC CCL-61TM), Led [originally named Pro-5WgaRI3C] (ATCC CRL- 1735TM), Pro-5 (ATCC CRL-1781TM), ACY1 -E (ATCCACC), ATCC
  • CHO cell lines include, e.g., FREESTYLETM CHO-S Cells and FLP-INTM-CHO Cell Line from Life Technologies.
  • the recombinant nucleic acids are codon optimized or codon-pair optimized for expression in a selected prokaryotic or eukaryotic host cell.
  • the nucleic acids can be incorporated into a vector, such as a prokaryotic or a eukaryotic expression vector.
  • exemplary vectors include plasmids that are able to replicate autonomously or to be replicated in a host cell.
  • Typical expression vectors contain suitable promoters, enhancers, and terminators that are useful for regulation of the expression of the coding sequence(s) in the expression construct.
  • the vectors may also comprise selection markers to provide a phenotypic trait for selection of transformed host cells (such as conferring resistance to antibiotics such as ampicillin or neomycin).
  • Nucleic acid or vector modification may be undertaken in a manner known by the art, see e.g., WO 2012/049317 (corresponding to US 2013/0216613) and WO 2016/092460 (corresponding to US 2018/0265551).
  • the nucleic acids that encode a modified HCMV gB protein as described herein are cloned into a vector suitable for introduction into mammalian cells (e.g., CHO cells).
  • the polynucleotide sequence that encodes the modified HCMV gB protein is introduced into the pMax vector developed by Amaxa.
  • the polypeptide is expressed under a constitutive promoter, the immediate early CMV promoter.
  • Selection of the stably transfected cells expressing the polypeptide is made based on the ability of the transfected cells to grow in the presence of kanamycin.
  • Cells that have successfully integrated the pMax are able to grow in the presence of kanamycin, because the pMax vector expresses a kanamycin resistance gene.
  • Selected cells can be clonally expanded and characterized for expression of the modified HCMV gB proteins.
  • the polynucleotide sequences that encode the modified gB proteins of the invention may be introduced into the pTT5 vector developed by NRC, which expresses an ampicillin resistance gene.
  • the expressed polypeptides are recovered (e.g., purified, isolated, or enriched).
  • the modified HCMV gB protein(s) may include a C- terminal polyhistidine tag (e.g., 6xHis tag) and, optionally, further include a cleavage site (e.g., a TEV cleavage site).
  • purification refers to the process of removing components from a composition or host cell or culture, the presence of which is not desired. Purification is a relative term and does not require that all traces of the undesirable component be removed from the composition. In the context of vaccine production, purification includes such processes as centrifugation, dialyzation, ion-exchange chromatography, and size-exclusion chromatography, affinity-purification or precipitation. Thus, the term“purified” does not require absolute purity; rather, it is intended as a relative term.
  • a preparation of substantially pure nucleic acid or protein can be purified such that the desired nucleic acid, or protein, represents at least 50% of the total nucleic acid content of the preparation.
  • a substantially pure nucleic acid, or protein will represent at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the total nucleic acid or protein content of the preparation.
  • Immunogenic molecules or antigens or antibodies which have not been subjected to any purification steps (i.e., the molecule as it is found in nature) are not suitable for pharmaceutical (e.g., vaccine) use.
  • a“purified” or an“isolated” biological component (such as a nucleic acid molecule, or protein) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs or was produced, such as, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been“isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.
  • the modified HCMV gB protein (or immunogenic fragment thereof) described herein is purified or isolated.
  • the modified gB protein (or immunogenic fragment thereof) can be purified using any suitable methods, such as HPLC, various types of chromatography (such as hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion), electrophoresis, density gradient centrifugation, solvent extraction, or the like.
  • HPLC high-denste
  • various types of chromatography such as hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion
  • electrophoresis electrophoresis
  • density gradient centrifugation solvent extraction, or the like.
  • methods for purifying HCMV gB protein by immunoaffinity chromatography has been disclosed. Ruiz-Arguello et al. , J. Gen. Virol., 85:3677-3687 (2004).
  • the modified gB protein may be further purified, as required, so as to remove substantially any proteins which are also secreted in the medium or result from lysis of host cells, so as to provide a product which is at least substantially free of host debris, e.g., proteins, lipids and polysaccharides. See, e.g., those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al.
  • the modified gB protein (or immunogenic fragment thereof) can include a“tag” that facilitates purification, as described above.
  • Recombinant as used herein to describe a polynucleotide means a polynucleotide of genomic, cDNA, RNA (including mRNA) semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • A“heterologous” sequence with regard to a HCMV gB protein refers to an amino acid or nucleotide sequence that is not found in naturally occurring HCMV gB protein, or in a nucleic acid encoding an HCMV gB protein, respectively.
  • the nucleic acid molecule may be referred to as “heterologous” (i.e. , the nucleic acid molecule is heterologous to at least the polynucleotide).
  • the polypeptide when a polypeptide is in contact with or in a complex with another protein that it is not associated with in nature, the polypeptide may be referred to as“heterologous” (i.e., the polypeptide is heterologous to the protein).
  • the nucleic acid molecule and polypeptide when a host cell comprises a nucleic acid molecule or polypeptide that it does not naturally comprise, the nucleic acid molecule and polypeptide may be referred to as“heterologous” (i.e., the nucleic acid molecule is heterologous to the host cell and the polypeptide is heterologous to the host cell).
  • sequence identity is frequently measured in terms of percentage identity (or similarity); the higher the percentage, the more similar are the primary structures of the two sequences. In general, the more similar the primary structures of two amino acid (or polynucleotide) sequences, the more similar are the higher order structures resulting from folding and assembly.
  • Variants of HCMV gB proteins and polynucleotide sequences can have one or a small number of amino acid deletions, additions or substitutions as compared to another, but will nonetheless share a very high percentage of their amino acid, and generally their polynucleotide sequence.
  • Methods of determining sequence identity are well known in the art.
  • Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151 , 1989; Corpet et al.
  • NCBI Basic Local Alignment Search Tool (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • This algorithm is conveniently implemented in the needle tool in the EMBOSS package (Rice et al., EMBOSS: The European Molecular Biology Open Software Suite, 2000 Trends Genetics 16: 276- 277). Sequence identity should be calculated over the entire length of the polypeptide sequence ofthe invention.
  • A“subject” is a living multi-cellular vertebrate organism.
  • the subject can be an experimental subject, such as a non-human mammal, e.g., a mouse, a cotton rat, or a non-human primate.
  • the subject can be a human subject.
  • an “antigen” is a compound, composition, or substance that can stimulate an immune response by producing antibodies and/or a T cell response in a mammal, including compositions that are injected, absorbed or otherwise introduced into a mammal.
  • the term“antigen” includes all related antigenic epitopes.
  • the term“epitope” or“antigenic determinant” refers to a site on an antigen to which B and/orT cells respond.
  • The“predominant antigenic epitopes” are those epitopes to which a functionally significant host immune response, e.g., an antibody response or a T-cell response, is made.
  • the predominant antigenic epitopes are those antigenic moieties that when recognized by the host immune system result in protection from disease caused by the pathogen.
  • the term“T-cell epitope” refers to an epitope that when bound to an appropriate MHC molecule is specifically bound by a T cell (via a T cell receptor).
  • A“B-cell epitope” is an epitope that is specifically bound by an antibody (or B cell receptor molecule).
  • An‘‘immune response” is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies.
  • An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response.
  • the response is specific for a particular antigen (that is, an‘antigen-specific response”), such as an HCMV antigen.
  • the antigen-specific response is a ‘‘pathogen-specific response.”
  • A‘‘protective immune response” is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen.
  • a protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen challenge in vivo.
  • the immune response elicits an immune response characterized by the production of Th1 type cytokines, e.g. a Th1-type immune response.
  • Th1 type cytokines e.g. a Th1-type immune response.
  • A“Th1” type immune response is characterized CD4+ T helper cells that produce IL-2 and IFN-y.
  • a“Th2” type immune response is characterized by CD4+ helper cells that produce IL-4, IL-5, and IL-13.
  • an “effective amount” means an amount sufficient to cause the referenced effect or outcome.
  • An “effective amount” can be determined empirically and in a routine manner using known techniques in relation to the stated purpose.
  • An‘‘immunologically effective amount” is a quantity of a composition (typically, an immunogenic composition) sufficient to elicit an immune response in a subject (either in a single dose or in a series).
  • the desired result is the production of an antigen (e.g., pathogen)-specific immune response that is capable of or contributes to protecting the subject against the pathogen, such as HCMV.
  • an antigen e.g., pathogen
  • the term immunologically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a protective immune response.
  • immunogenic compositions or pharmaceutical compositions such as vaccines, that comprise a modified HCMV gB protein and a pharmaceutically acceptable diluent, carrier or excipient.
  • An‘‘immunogenic composition” is a pharmaceutical composition of matter suitable for administration to a human or non-human mammalian subject that is capable of eliciting a specific immune response, e.g., against a pathogen, such as HCMV.
  • an immunogenic composition includes one or more antigens (for example, polypeptide antigens or an RNA or DNA that encodes a polypeptide antigen) or antigenic epitopes, such as for instance, the modified HCMV gB protein of the inventions.
  • An immunogenic composition can also include one or more additional components capable of enhancing an immune response, such as an excipient, carrier, and/or adjuvant.
  • immunogenic compositions are administered to elicit an immune response that protects the subject against symptoms or conditions induced by a pathogen.
  • symptoms or disease caused by a pathogen is prevented (or reduced or ameliorated) by inhibiting replication of the pathogen (e.g. , HCMV) following exposure of the subject to the pathogen.
  • pathogen e.g. , HCMV
  • the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against HCMV.
  • the immunogenic compositions according to the invention are not limited to compositions consisting of modified HCMV gB proteins.
  • the present invention also contemplates immunogenic compositions, such as vaccines, comprising the modified HCMV gB protein of the invention and at least one or more HCMV antigens.
  • Such one or more HCMV antigens may be pp65, IE1 , gO, gl_, gH, pUL128, pUL130, pUL131 , or any combination thereof, or any complex-forming fragment thereof.
  • a composition comprising a modified HCMV gB protein as described herein and pp65 is contemplated.
  • compositions comprising a modified protein as described herein and IE1 are contemplated .
  • a composition comprising a modified HCMV gB protein as described herein and gl_, gH, pUL128, pUL130, and pUL131 is contemplated.
  • compositions herein may comprise one or more nucleic acids (e.g., an RNA) that together encode a modified HCMV gB protein as described herein and one or more of a gO, gl_, gH, pUL128, pUL130, pUL131 , pp65, or IE1 protein (e.g., encode a HCMV gO, gL, gH, pUL128, pUL130, pUL131 , pp65, or IE1 protein).
  • nucleic acids e.g., an RNA
  • IE1 protein e.g., encode a HCMV gO, gL, gH, pUL128, pUL130, pUL131 , pp65, or IE1 protein.
  • a“complex-forming fragment’’ it is meant any part or portion of the polypeptide that retains the ability to form a complex (e.g ., the pentameric complex, gH/gL dimer, and gH/gL/gO trimer) with other polypeptides of the complex.
  • a“complex-forming fragment’’ of a modified protein comprises the one or more modified amino acid residues (i.e., the fragment of a modified protein comprises the modification(s)).
  • composition comprises gL, gH, pUL128, pUL130, or pUL131 (five proteins that together form what is called a“pentamer complex’’)
  • a“pentamer-forming’’ fragment of gL, gH, pUL128, pUL130, or pUL131 may be utilized (see WO 2014/005959 (corresponding to US 9.683,022); WO2016/1 16904 (corresponding to US 10,167,321); and WO 2018/193307).
  • the composition comprises gH (e.g., HCMV gH)
  • the gH may be a complex-forming fragment that lacks a transmembrane domain (see WO 2014/005959 (corresponding to US 9.683,022).
  • the composition comprises gL (e.g., HCMV gL), the gL may be a modified gL protein having a modification within a protease recognition site, e.g., a modification within a protease recognition site that reduces protease cleavage of gL (see WO2016/1 16904 (corresponding to US 10,167,321)).
  • the composition comprises one or more HCMV gL, gH, pUL128, pUL130, or pUL131 proteins; such one or more HCMV proteins may comprise one or more stabilizing modification as previously disclosed within WO 2018/193307.
  • diluents and carriers and/or pharmaceutically acceptable excipients are known in the art and are described, e.g., in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975).
  • pharmaceutically acceptable indicates that the diluent, or carrier, or excipient, is suitable for administration to a subject (e.g., a human or non-human mammalian subject).
  • a subject e.g., a human or non-human mammalian subject.
  • the nature of the diluent, carrier and/or excipient will depend on the particular mode of administration being employed.
  • parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like.
  • a liquid diluent is not employed.
  • non-toxic solid carriers can be used, including for example, pharmaceutical grades of trehalose, mannitol, lactose, starch or magnesium stearate.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins (e.g., nanoparticles), polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • proteins e.g., nanoparticles
  • polysaccharides e.g., polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • suitable excipients and carriers can be selected by those of skill in the art to produce a formulation suitable for delivery to a subject by a selected route of administration.
  • Excipients include, without limitation: glycerol, polyethylene glycol (PEG), glass forming polyols (such as, sorbitol, trehalose) N-lauroylsarcosine (e.g., sodium salt), L -proline, non detergent sulfobetaine, guanidine hydrochloride, urea, trimethylamine oxide, KCI, Ca2+, Mg2+ , Mn2+ , Zn2+ (and other divalent cation related salts), dithiothreitol (DTT), dithioerytrol, b-mercaptoethanol, detergents (including, e.g., TWEEN 80, TWEEN 20, TRITON X-100, NP-40, EMPIGEN BB, Octylglucoside, Lauroyl malto
  • the antigen(s) may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, or another pathogen.
  • a bacterial toxoid such as a toxoid from diphtheria, tetanus, cholera, H. pylori, or another pathogen.
  • the modified HCMV gB protein is operably linked to a carrier wherein the carrier is a nanoparticle.
  • Pharmaceutically acceptable nanoparticles as carriers as well as methods of using them to present an antigen are known and include ferritin or lumazine synthase nanoparticles or nanoparticles derived therefrom (see WO 2005/121330 (corresponding to US 8,685,670); WO 2013/044203 (corresponding to US 10,137,190); and WO 2016/037154).
  • Such nanoparticles may be“selfassembling’’ (see WO 2015/048149 (corresponding to US 2015/0110825)).
  • Nanoparticles operably linked to an antigen in pre-fusion conformation has been demonstrated for RSV F proteins (US 9,738,689).
  • the pharmaceutical formulations are conventionally administered parenterally, e.g., by injection, either subcutaneously, intraperitoneally, transdermally, or intramuscularly. Some embodiments will be administered through an intra-mucosal route such as intra-orally, intra-nasally, intra-vag inally , and intra-rectally. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral formulations may be preferred for certain viral proteins. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the immunogenic composition may be administered in conjunction with other immunoregulatory agents. Any suitable route of administration can be used.
  • a composition can be administered intramuscularly, intraperitoneally, subcutaneously, or transdermally.
  • Compositions can be administered according to any suitable schedule.
  • the pharmaceutical formulations are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
  • the immunogenic composition also includes an adjuvant.
  • Suitable adjuvants for use in immunogenic compositions containing modified HCMV gB proteins of the invention are adjuvants that in combination with said polypeptides disclosed herein are safe and having acceptable reactogenicity when administered to a subject.
  • An“adjuvant” is an agent that enhances the production of an immune response in a nonspecific manner.
  • Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) onto which antigen may be adsorbed; emulsions, including water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists), and various combinations of such components.
  • the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod.
  • the adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate.
  • the adjuvants described herein can be used singularly or in any combination, such as alum/TLR7 agonist combination.
  • Suitable adjuvants for use in combination with the modified gB proteins of the invention are saponins.
  • immunogenic compositions of the invention may comprise the saponin QS21 (WO8809336A1 ; US5057540A).
  • QS21 is well known in the art as a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant lgG2a antibody response.
  • CTLs cytotoxic T cells
  • Th1 cells a predominant lgG2a antibody response.
  • OPT-821 cytotoxic T cells
  • the immunogenic compositions of the invention comprise QS21 in substantially pure form, that is to say, the QS21 is at least 80% , at least 85%, at least 90% pure, for example at least 95% pure, or at least 98% pure.
  • the immunogenic compositions comprising the modified HCMV gB proteins of the invention may comprise QS21 and a sterol, cholesterol in particular. Such compositions show a decreased reactogenicity when compared to compositions in which the sterol is absent, while the adjuvant effect is maintained. Reactogenicity studies may be assessed according to the methods disclosed in WO 96/33739. Suitably the sterol is associated to the saponin adjuvant as described in WO 96/33739.
  • the cholesterol is present in excess to that of QS21 , for example, the ratio of QS21 :sterol will typically be in the order of 1 : 100 to 1 :1 (w/w), suitably between 1 :10 to 1 :1 (w/w), and preferably 1 :5 to 1 : 1 (w/w). In particular, the ratio of QS21 :sterol being at least 1 :2 (w/w). In a particular embodiment, the ratio of QS21 :sterol is 1 :5 (w/w). Suitable sterols include b-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol.
  • compositions of the invention comprise cholesterol as sterol.
  • sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 1 1th Edn, page 341 , as a naturally occurring sterol found in animal fat.
  • immunogenic compositions comprising the modified modified gB proteins of the invention comprise QS21 in its less reactogenic composition where it is quenched with a sterol, such as cholesterol for example.
  • a sterol such as cholesterol for example.
  • the saponin /sterol is in the form of a liposome structure (WO 96/337391).
  • modified HCMV gB proteins of the invention can suitably be employed in immunogenic compositions with an adjuvant comprising a combination of QS21 and cholesterol.
  • Nposome(s) generally refers to uni- or multilamellar (particularly 2, 3, 4, 5, 6, 7, 8, 9, or 10 lamellar depending on the number of lipid membranes formed) lipid structures enclosing an aqueous interior. Liposomes and liposome formulations are well known in the art. Lipids, which are capable of forming liposomes include all substances having fatty or fat-like properties.
  • Lipids which can make up the lipids in the liposomes can be selected from the group comprising of glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines, sterols, archeolipids, synthetic cationic lipids and carbohydrate containing lipids.
  • Liposomes may suitably comprise a phospholipid.
  • Suitable phospholipids include (but are not limited to): phosphocholine (PC) which is an intermediate in the synthesis of phosphatidylcholine; natural phospholipid derivates: egg phosphocholine, egg phosphocholine, soy phosphocholine, hydrogenated soy phosphocholine, sphingomyelin as natural phospholipids; and synthetic phospholipid derivates: phosphocholine (didecanoyl-L-a-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoyl phosphatidylcholine [DPPC], distearoyl phosphatidylcholine [DSPC], dioleoyl phosphatidylcholine [DOPC], 1 -palmitoyl, 2- oleoylphosphatidylcholine [POPC], dielaidoyl phosphatidylcholine
  • the liposomes comprise 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine.
  • highly purified phosphatidylcholine is used and can be selected from the group comprising phosphatidylcholine (Egg), phosphatidylcholine hydrogenated (Egg) phosphatidylcholine (Soy) phosphatidylcholine hydrogenated (Soy).
  • the liposomes comprise phosphatidylethanolamine [POPE] or a derivative thereof. Liposome size may vary from 30 nm to several pm depending on the phospholipid composition and the method used for their preparation.
  • the liposome size will be in the range of 50 nm to 500 nm and in further embodiments 50 nm to 200 nm. Dynamic laser light scattering is a method used to measure the size of liposomes well known to those skilled in the art.
  • Liposomes of the invention may comprise dioleoyl phosphatidylcholine [DOPC] and a sterol, in particular cholesterol.
  • immunogenic compositions comprising the modified HCMV gB proteins of the invention, comprise QS21 in the form of a liposome, wherein said liposome comprises dioleoyl phosphatidylcholine [DOPC] and a sterol, in particular cholesterol.
  • Immunogenic compositions of the invention may comprise one or more further immunostimulants.
  • immunogenic compositions comprising the modified HCMV gB proteins of the invention as described herein further comprise a lipopolysaccharide, suitably a non-toxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly 3- Deacylated monophoshoryl lipid A (3D - MPL).
  • 3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals and is referred throughout the specification as MPL or 3D-MPL. See, for example, US Patent Nos. 4,436,727; 4,877,61 1 ; 4,866,034 and 4,912,094.
  • 3D-MPL primarily promotes CD4+ T cell responses with an IFN- g (Th1) phenotype.
  • 3D-MPL can be produced according to the methods disclosed in GB2220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains.
  • small particle 3D-MPL can be used. Small particle 3D-MPL has a particle size such that it can be sterile-filtered through a 0.22m ⁇ ti filter. Such preparations are described in W094/21292.
  • the lipopolysaccharide can be a b1 -6) glucosamine disaccharide, as described in US Patent No. 6,005,099 and EP Patent No. 0 729 473 B1.
  • a lipopolysaccharide such as 3D-MPL
  • acylated monosaccharide and disaccharide derivatives that are a sub-portion to the above structure of MPL are also suitable adjuvants.
  • the adjuvant is a synthetic derivative of lipid A, some of which are described as TLR-4 agonists ( see WO 95/14026; WO 99/64301 and WO 00/0462; WO 01/46127).
  • compositions with modified HCMV gB proteins can also be used in compositions with modified HCMV gB proteins.
  • QS21 can be formulated together with 3D-MPL.
  • the ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1 ; such as 1 :5 to 5 : 1 , and often substantially 1 : 1.
  • the ratio is in the range of 2.5 : 1 to 1 : 1 3D-MPL: QS21.
  • immunogenic compositions comprising modified gB proteins of the invention comprise at least QS21 and 3D-MPL.
  • the immunogenic compositions comprising the modified HCMV gB proteins of the invention may also be suitably formulated with an oil-in-water emulsion.
  • the oil in water emulsion comprises a metabolisable oil (i.e. biodegradable).
  • the oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also suitable. Accordingly, oil-in-water emulsions used in combination with the modified HCMV gB proteins of the invention comprise a metabolisable oil.
  • oil- in-water emulsions comprise squalene (for example between about 4% and 6% [v/v]).
  • the oil-in-water emulsion may further comprise a surfactant.
  • Oil-in-water emulsions of the invention comprise one or more surfactants. Suitable surfactants are well known to the skilled person and include, but are not limited to, polyoxyethylene sorbitan monooleate (TWEEN 80, Polysorbate 80), sorbitan triolate (SPAN 85), phosphatidylcholine (lecithin), polyoxyethylene (12) cetostearyl ether and octoxynol-9 (TRITON X- 100).
  • oil-in-water emulsions comprise is polyoxyethylene sorbitan monooleate (TWEEN 80, Polysorbate 80).
  • oil in water emulsions of the invention comprise polyoxyethylene sorbitan monooleate (TWEEN 80) and a further surfactant, in particular sorbitan trioleate (SPAN 85).
  • Oil-in-water emulsions of the invention may also comprise a tocol.
  • Tocols are well known in the art and are described in EP0382271 .
  • the tocol is a-tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate).
  • immunogenic compositions comprising the modified gB protein of the invention in combination with an oil-in-water emulsion comprising squalene (for example about 5% [v/v]) and a- tocopherol (for example about 5% [v/v]).
  • the oil-in-water emulsion comprises a metabolisable oil (e.g. squalene), a tocol (e.g. a-tocopherol) and a surfactant (e.g. polyoxyethylene sorbitan monooleate [Polysorbate 80]).
  • oil-in-water emulsions of the invention comprise a metabolisable oil (e.g. squalene), a surfactant (e.g. polyoxyethylene sorbitan monooleate [Polysorbate 80]), and optionally a second surfactant (e.g. sorbitan trioleate [SPAN 85]).
  • oil-in-water emulsions of the invention comprise a metabolisable oil (e.g. squalene), a polyoxyethylene alkyl ether hydrophilic non-ionic surfactant (e.g.
  • immunogenic compositions comprise an oil-in-water emulsion comprising squalene, alpha-tocopherol, and Polysorbate 80.
  • the oil-in-water comprises 11 mg metabolisable oil (such as squalene) or below, for example between 0.5-11 mg, 0.5-10 mg or 0.5-9 mg 1 -10 mg, 1-1 1 mg, 2-10 mg, 4-8 mg, or 4.5- 5.5 mg, and 5 mg emulsifying agent (such as polyoxyethylene sorbitan monooleate) or below, for example between 0.1-5 mg, 0.2-5 mg, 0.3-5 mg, 0.4-5 mg, 0.5-4 mg, 1-2 mg or 2-3 mg per dose of the vaccine.
  • tocol e.g.
  • alpha-tocopherol where present is 12 mg or below, for example between 0.5-12 mg, 10-11 mg, 1-11 mg, 2-10 mg, 4-9 mg, or 5-7 mg per human vaccine dose.
  • a human dose is meant a dose which is in a volume suitable for human use. Generally, this is between 0.25 and 1.5 ml.
  • a human dose is 0.5 ml.
  • a human dose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml.
  • a human dose is between 1 ml and 1 .5 ml.
  • a human dose may be less than 0.5 ml such as between 0.25 and 0.5 ml.
  • An immunogenic composition typically contains an immunoprotective quantity (or a fractional dose thereof) of the antigen and can be prepared by conventional techniques.
  • Preparation of immunogenic compositions, such as vaccines, including those for administration to human subjects, is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design-the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al. , University Park Press, Baltimore, Maryland, U.S.A. 1978.
  • Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877.
  • Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S.
  • the amount of protein in each dose of the immunogenic composition is selected as an amount which induces an immunoprotective response without significant, adverse side effects in the typical subject. Immunoprotective in this context does not necessarily mean completely protective against infection; it means protection against symptoms or disease, especially severe disease associated with the virus.
  • the amount of antigen can vary depending upon which specific immunogen is employed. The amount utilized in an immunogenic composition is selected based on the subject population (e.g., infant or elderly). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects can receive a boost in about four weeks.
  • modified FICMV gB proteins (or immunogenic fragments thereof) and/or one or more additional immunogenic protein (e.g., FICMV antigen) described herein are delivered using alphavirus replicon particles (VRP).
  • VRP alphavirus replicon particles
  • the term“alphavirus’’ has its conventional meaning in the art and includes various species such as Venezuelan equine encephalitis virus (VEE; e.g., Trinidad donkey, TC83CR, etc.), Semliki Forest virus (SFV), Sindbis virus, Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, S.A.
  • AR86 virus Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.
  • VRP alphavirus replicon particle
  • replicon particle is an alphavirus replicon packaged with alphavirus structural proteins.
  • An ‘‘alphavirus replicon” is an RNA molecule which can direct its own amplification in vivo in a target cell.
  • the replicon encodes the polymerase(s) which catalyze RNA amplification (nsPI, nsP2, nsP3, nsP4) and contains cis RNA sequences required for replication which are recognized and utilized by the encoded polymerase(s).
  • An alphavirus replicon typically contains the following ordered elements: 5’ viral sequences required in cis for replication, sequences which encode biologically active alphavirus nonstructural proteins (nsPI, nsP2, nsP3, nsP4), 3’ viral sequences required in cis for replication, and a polyadenylate tract.
  • An alphavirus replicon also may contain one or more viral subgenomic‘‘junction region” promoters directing the expression of heterologous nucleotide sequences, which may, in certain embodiments, be modified in order to increase or reduce viral transcription of the subgenomic fragment and heterologous sequence(s) to be expressed.
  • Other control elements can be used, such as IRES or 2A sequences.
  • Recombinant nucleic acids that encode the modified HCMV gB proteins or immunogenic fragments described herein can be administered to induce production of the encoded modified gB proteins or immunogenic fragments and an immune response thereto.
  • the recombinant nucleic acid can be DNA (e.g., plasmid or viral DNA) or RNA, preferably self-replicating RNA, and can be monocistronic or polycistronic. Any suitable DNA or RNA can be used as the nucleic acid vectorthat carries the open reading frames that encode HCMV gB proteins or immunogenic fragments thereof. Suitable nucleic acid vectors have the capacity to carry and drive expression of one or more modified HCMV gB proteins or immunogenic fragments.
  • nucleic acid vectors include, for example, plasmids, DNA obtained from DNA viruses such as vaccinia virus vectors (e.g., NYVAC, see US 5,494,807), adenoviral vectors and poxvirus vectors (e.g., ALVAC canarypox vector, Sanofi Pasteur), and RNA obtained from suitable RNA viruses such as alphavirus.
  • DNA viruses such as vaccinia virus vectors (e.g., NYVAC, see US 5,494,807)
  • adenoviral vectors and poxvirus vectors e.g., ALVAC canarypox vector, Sanofi Pasteur
  • RNA obtained from suitable RNA viruses such as alphavirus.
  • the recombinant nucleic acid molecule can be modified, e.g., contain modified nucleobases and or linkages as described further herein.
  • the self-replicating RNA molecules of the invention are based on the genomic RNA of RNA viruses, but lack the genes encoding one or more structural proteins.
  • the self-replicating RNA molecules are capable of being translated to produce non-structural proteins of the RNA virus and modified HCMV gB proteins encoded by the self-replicating RNA.
  • the self-replicating RNA generally contains at least one or more genes selected from the group consisting of viral replicase, viral proteases, viral helicases and other nonstructural viral proteins, and also comprise 5'- and 3'-end cis-active replication sequences, and a heterologous sequence that encodes one or more desired modified HCMV gB proteins.
  • a subgenomic promoter that directs expression of the heterologous sequence(s) can be included in the self-replicating RNA.
  • a heterologous sequence may be fused in frame to other coding regions in the self- replicating RNA and/or may be under the control of an internal ribosome entry site (IRES).
  • Self-replicating RNA molecules of the invention can be designed so that the self-replicating RNA molecule cannot induce production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary for the production of viral particles in the self-replicating RNA.
  • an alpha virus such as Sindbis virus (SIN), Semliki forest virus and Venezuelan equine encephalitis virus (VEE)
  • Sindbis virus Sindbis virus
  • VEE Venezuelan equine encephalitis virus
  • one or more genes encoding viral structural proteins, such as capsid and/or envelope glycoproteins can be omitted.
  • self-replicating RNA molecules of the invention can be designed to induce production of infectious viral particles that are attenuated or virulent, orto produce viral particles that are capable of a single round of subsequent infection.
  • a self-replicating RNA molecule can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (or from an antisense copy of itself).
  • the self-replicating RNA can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These transcripts are antisense relative to the delivered RNA and may be translated themselves to provide in situ expression of encoded HCMV protein, or may be transcribed to provide furthertranscripts with the same sense as the delivered RNA which are translated to provide in situ expression of the encoded HCMV protein(s).
  • a preferred self-replicating RNA molecule thus encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) one or more modified HCMV gB proteins or immunogenic fragments thereof.
  • the polymerase can be an alphavirus replicase e.g. comprising alphavirus non-structural proteins nsP1-nsP4.
  • the self-replicating RNA molecules of the invention can contain one or more modified nucleotides and therefore have improved stability and be resistant to degradation and clearance in vivo, and other advantages.
  • modified nucleoside modifications found on mammalian RNA. See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994).
  • the preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, e.g.
  • the self-replicating RNA molecule can contain phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.
  • the self-replicating RNA described herein is suitable for delivery in a variety of modalities, such as naked RNA delivery or in combination with lipids, polymers or other compounds that facilitate entry into the cells.
  • Self-replicating RNA molecules can be introduced into target cells or subjects using any suitable technique, e.g., by direct injection, microinjection, electroporation, lipofection, biolistics, and the like.
  • the self-replicating RNA molecule may also be introduced into cells by way of receptor-mediated endocytosis. See e.g., U.S. Pat. No. 6,090,619; Wu and Wu, J. Biol. Chem., 263:14621 (1988); and Curiel et al distribute Proc. Natl. Acad. Sci. USA, 88:8850 (1991).
  • 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety (e.g., poly-L-lysine having 3-100 lysine amino acids), which is itself coupled to an integrin receptor-binding moiety (e.g., a cyclic peptide having the sequence Arg-Gly-Asp).
  • a polycation moiety e.g., poly-L-lysine having 3-100 lysine amino acids
  • an integrin receptor-binding moiety e.g., a cyclic peptide having the sequence Arg-Gly-Asp
  • the self-replicating RNA molecules can be delivered into cells via amphiphiles. See e.g., U.S. Pat. No. 6,071 ,890.
  • a nucleic acid molecule may form a complex with the cationic amphiphile. Mammalian cells contacted with the complex can readily take it up.
  • the self-replicating RNA can be delivered as naked RNA (e.g. merely as an aqueous solution of RNA) but, to enhance entry into cells and also subsequent intercellular effects, the self-replicating RNA is preferably administered in combination with a delivery system, such as a particulate or emulsion delivery system.
  • a delivery system such as a particulate or emulsion delivery system.
  • a large number of delivery systems are well known to those of skill in the art.
  • Three particularly useful delivery systems are (i) liposomes, (ii) non-toxic and biodegradable polymer microparticles, and (iii) cationic submicron oil-in-water emulsions.
  • the invention also provides immunogenic composition
  • the nucleic acid e.g., self- replicating RNA
  • the immunogenic composition may comprise an adjuvant, as described above.
  • Preferred adjuvants include, e.g., an aluminum salt or an oil-in-water emulsion (such as MF59).
  • Methods of inducing an immune response against FICMV may comprise the production of neutralizing antibodies against FICMV.
  • the neutralizing antibodies are complement-independent.
  • the induced immune response can comprise a humoral immune response, a cell-mediated immune response, or both.
  • an immune response is induced against each delivered FICMV gB protein and, as relevant, other virus antigens having been delivered (e.g., pentamer complex).
  • a cell-mediated immune response can comprise a helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both.
  • the induced immune response comprises a humoral immune response, and the antibodies are neutralizing antibodies.
  • Neutralizing antibodies block viral infection of cells.
  • the FICMV infects epithelial cells and also fibroblast cells and, in some embodiments the immune response reduces or prevents HCMV infection of both cell types.
  • Neutralizing antibody responses can be complement-dependent or complement-independent.
  • the induced neutralizing antibody response is complement-dependent.
  • the induced neutralizing antibody response is cross-neutralizing; e.g., with regard to HCMV, an antibody generated against an administered composition neutralizes a HCMV virus of one or more strains in addition to the strain used in the composition.
  • An immune response can be stimulated by administering proteins, DNA molecules, RNA molecules (e.g., self-replicating RNA molecules), or VRPs to an individual subject, typically a mammal, including a human.
  • the immune response induced is a protective immune response, i.e. , the response reduces the risk or severity of HCMV infection.
  • Stimulating a protective immune response is particularly desirable in populations particularly at risk for HCMV infection, including solid organ transplant (SOT) patients, bone marrow transplant patients, and hematopoietic stem cell transplant (HSCT) patients.
  • VRPs can be administered to a transplant donor pre-transplant, or a transplant recipient pre- and/or post-transplant. Because vertical transmission of HCMV from motherto child is a common source of infection in infants, administering VRPs to a woman who is pregnant or can become pregnant is particularly useful.
  • the invention provides a kit for identifying the presence of a modified HCMV gB protein within a composition, for the purification of a modified HCMV gB protein from a composition, or for detecting a modified HCMV gB protein in pre-fusion or non-post-fusion conformation; the kit comprising an antibody or antigen-binding fragment thereof that is specific for the modified HCMV gB protein.
  • the invention further provides a pharmaceutical kit for the prevention or treatment of HCMV virus infection comprising a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof that is specific for a modified HCMV gB protein as described herein.
  • the kit also contains instructions for using the antibody or antigen-binding fragment thereof for identifying the presence of a modified HCMV gB protein within a composition, for the purification of a modified HCMV gB protein from a composition, or for the prevention or treatment of HCMV infection.
  • the kit may also contain excipients, diluents, adjuvants, syringes, other appropriate means of administering the antibody or antigen-binding fragment thereof, or instructions for decontamination or disposal.
  • gB protein or polynucleotide is‘‘truncated” herein it is generally meant that the gB protein or polynucleotide is truncated as compared to a wild type or control gB protein or polynucleotide (i.e., it is the native amino acids or nucleic acids, found within a wild type gB protein or polynucleotide, which have been deleted).
  • ‘Operably linked” means connected so as to be‘‘operational”, for example, the configuration of polynucleotide sequences for recombinant protein expression.
  • ‘‘operably linked” refers to the art-recognized positioning of, e.g., nucleic acid components such that the intended function (e.g., expression) is achieved.
  • nucleic acid components such that the intended function (e.g., expression) is achieved.
  • two or more components "operably linked” together are not necessarily adjacent to each other in the nucleic acid or amino acid sequence.
  • a coding sequence that is ‘‘operably linked” to a ‘‘control sequence” e.g., a promoter, enhancer, or IRES
  • a ‘control sequence” e.g., a promoter, enhancer, or IRES
  • heterologous sequence must be operably linked to the native HCMV gB amino acids (likewise the polynucleotide sequence encoding the heterologous sequence must be operably linked to the polynucleotide sequence encoding the native HCMV gB amino acids).
  • the heterologous sequence must be operably linked to the native HCMV gB amino acids (likewise the polynucleotide sequence encoding the heterologous sequence must be operably linked to the polynucleotide sequence encoding the native HCMV gB amino acids).
  • “between about 5.5 to 6.5 mg/ml” means the boundaries of the numerical range extend below 5.5 and above 6.5 so that the particular value in question achieves the same functional result as within the range.
  • “about” and “approximately” can mean within one or more than one standard deviation as per the practice in the art.
  • “about” and “approximately” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1 % of a given value.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • steps of a method may be numbered (such as (1), (2), (3), etc. or (i), (ii), (iii)), the numbering of the steps does not mean that the steps must be performed in that order (i.e., step 1 then step 2 then step 3, etc.).
  • the word“then” may be used to specify the order of a method’s steps.
  • HCMV Merlin strain gB (SEQ ID NO: 1 and UniProtKB Accession No. F5HB53) was modified with previously described furin cleavage site modifications to reduce processing during protein expression (Chandramouli et al. 2015 Nat. Comm. 6(8176): 1-12; Burke & Heldwein 2015 PLOS Path. DOI: 10.1371 ; see also WO 2012/049317 (corresponding to US 2013/0216613); and WO 2016/092460 (corresponding to US 2018/0265551).
  • residue R457 (numbered perSEQ ID NO: 1) was substituted to serine (R457S) and residue R460 (numbered per SEQ ID NO: 1) was substituted to serine (R460S).
  • Fusion Loop 1 FL1
  • Fusion Loop 2 FL2
  • hydrophobic residues within FL1 or FL2 were replaced with their (generally) more hydrophilic FISV-1 counterparts (Burke & Fleldwein 2015 PLOS Path. DOI: 10.1371 ; see also VMO 2012/049317 (corresponding to US 2013/0216613); and WO 2016/092460 (corresponding to US 2018/0265551)).
  • substitutions were made (numbered per SEQ ID NO: 1) Y155G, I156H, H157R, W240A, L241 F, and Y242H.
  • a HCMV Merlin strain gB having such furin cleavage site and fusion loops modifications forms a trimer and has post-fusion conformation ( see Chandramouli et al. 2015 Nat. Comm. 6:8176).
  • FICMV Merlin strain gB molecules having the above furin cleavage site modifications (R457S and R460S) and the above FL1 and FL2 modifications (Y155G, I156H, H157R, W240A, L241 F, and Y242FI) are referred to herein as“gB’2” or‘gB prime 2’.
  • FICMV AD169 strain gB (SEQ ID NO: 6) was modified to contain the furin cleavage site modifications and fusion loop modifications corresponding to those in FICMV Merlin strain gB’2.
  • the resulting modified FICMV AD169 gB molecules are referred to herein as“gB’1” or‘gB prime one’.
  • the gB’2 molecule as described above was further modified to provide gB’-GCN4 (SEQ ID NO: 3) by: truncation of the residues C-terminal to MPR residue K724 (MPR corresponds to residues D699-P751 (inclusive) of SEQ ID NO: 1); insertion of two proline residues C-terminally adjacent to residue K724; and addition of the General Control Nonderepressible (GCN) 4 Leucine Zipperdomain in a trimeric oligomerization state (“GCN4” herein) having the sequence SEQ ID NO: 2 (see Protein Data Bank 4DME and Osschi etal. 2012 Biochem. 51 (47): 9581 -9591).
  • GCN4 General Control Nonderepressible
  • the GCN4 sequence is placed c-terminally adjacent to the c-terminal-most inserted proline residue.
  • This modified gB molecule is referred to herein as gB’-GCN4 (SEQ ID NO: 3).
  • Such a modified gB molecule can be represented by N’-K724-P-P-GCN4-C’.
  • gB’-GCN4 SEQ ID NO: 3 was further modified by operably linking a TEV-cleavage site and 6xHIS-tag (SEQ ID NO: 5) to the C-terminus of the GCN4 domain, to provide SEQ ID NO: 4 (gB’-GCN4-TEV).
  • the gB’1 , gB’2 and gB’-GCN4 constructs were made and expressed in transiently transfected Expi293 cells.
  • the presence of antigenic domain 5 (AD- 5) epitope in gB’1 , gB’2, and gB’-GCN4 was confirmed via binding to a neutralizing antibody (see Chandramouli et al. 2015 Nat. Comm. 6(8176): 1 -12 regarding AD-5; data not shown).
  • trimerization domain such as a GCN4 domain, C-terminally adjacent to the residue corresponding to K724 of SEQ ID NO: 1 , causes a mixed population ofgB molecules to be produced, including those in a post-fusion-like conformation (peak I) and those in a non-post-fusion conformation (peak II).
  • the GCN4 domain causes a gB molecule which would otherwise transition to post-fusion conformation (e.g., gB’1 , gB’2, or wild type gB), to reside in a non-post-fusion conformation.
  • the Domain III Coiled-coil region of FICMV gB spans amino acids N478 to I523 (inclusive) of SEQ ID NO: 1 (see Explanation of Sequences section below for a description of corresponding Dlllcc Region residues within SEQ ID NO: 6)
  • the analysis herein suggests that a proline substitution modification at one or more (e.g., one ortwo) of the amino acids N478-R511 (inclusive) of SEQ ID NO: 1 will stabilize the modified gB protein (gB’-Dlllcc-GCN4) in non-post-fusion conformation and will therefore drive host cell production towards the non-post- fusion gB conformation and away from the post-fusion gB conformation.
  • the ratio of non-post-fusion gB to post-fusion gB will increase when at least one proline substitution is made within the residues corresponding to N478-R51 1 (inclusive)of SEQ ID NO: 1 , in a gB molecule having a C-terminus truncation and addition of GCN4 domain as described herein (e.g., gB’-GCN4)).
  • gB’-R51 1 P-GCN4, gB’-R512P-GCN4, gB’-T513P-GCN4, gB’-L514P-GCN4, gB’-E515P-GCN4, gB’-V516P-GCN4, or gB’-F517P-GCN4 cells was about 0.5 (FIG. 4B). Therefore, the Q510P substitution being added to a gB-GCN4 construct as described herein increases the yield of gB molecules in non-post-fusion conformation (peak I I) by a factor of 1 .8 (FIG. 4B).
  • Additional gB’-GCN4 proline mutants were designed and evaluated for their effect on Peak l l/Peak I ratio.
  • A505P, W506P, C507P, D509P, D509P/Q510P double mutant and Q510P/R51 1 P double mutant (all numbered with respect to SEQ ID NO: 1 ) were designed .
  • Q510P/R51 1 P double mutant that was not expressed due to a believed contamination issue with the sample
  • all proteins were expressed in 30 ml of expression media, purified by single step purification using Nl SEPFIAROSE EXCEL (GE Flealthcare), or analyzed by NanoDSF and SEC-FIPLC.
  • W506P produced more Peak I I than any other construct.
  • A505P and C507P had Peak I I level similar to gB’-GCN4 (which is referred to as Wild Type (WT) in FIGs. 6A and 6B).
  • Peak II population for D509P and double mutant D509P/Q510P were slightly lower than Q510P (FIGs. 6A and 6B).
  • Another modified FICMV gB molecule was generated (referred to as“gB698-GCN4” herein) by further modifying the gB’2 molecule (described above) so that the entire MPR is deleted (i.e., residues D699-P751 of SEQ ID NO: 1 are deleted).
  • gB’-GCN4 described herein above comprises a truncated MPR (residues 699-724 of SEQ ID NO: 1) versus this gB698-GCN4 molecule has had the entire MPR deleted (both molecules are based on the FICMV Merlin strain gB).
  • gB698-GCN4 is characterized by having a truncation of the MPR residues D699-P751 (inclusive) of SEQ ID NO: 1 ; insertion of two proline residues C-terminally adjacent to residue E698 (i.e., P699 and P700 of SEQ ID NO: 11); and the c-terminal-most proline (P700) being operably linked to the GCN4 with sequence SEQ ID NO: 2.
  • gB698-GCN4 can be represented by N -E698-P- P-GCN4-C’.
  • a TEV cleavage site and 6xFlis tag (SEQ ID NO: 5) was inserted c-terminally adjacent to the GCN4. See the gB698-GCN4 amino acid sequence SEQ ID NO: 1 1.
  • EBV-GCN4 modified EBV gB molecule was generated (referred to as“EBV-GCN4” herein) by truncating the MPR (i.e., deleting the residues c-terminal to residue Q705); inserting two proline residues C-terminally adjacent to residue Q705 (i.e., P706 and P707 of SEQ ID NO: 13); and operably linking the c-terminal-most proline (P707) to the GCN4 with sequence SEQ ID NO: 2.
  • EBV-GCN4 can be represented by N’- Q705-P-P-GCN4-C’.
  • a TEV cleavage site and a 6xFlis tag (SEQ ID NO: 5) was inserted c-terminally adjacent to the GCN4. See the EBV-GCN4 amino acid sequence SEQ ID NO: 13.
  • gB698-GCN4 and EBV-GCN4 were expressed and purified as described above (expressed in 30 ml from EXPi293 mammalian cells and purified by Nl SEPHAROSE EXCEL (GE Healthcare) column).
  • gB698-GCN4 which does not contain an MPR, showed mainly Peak I under SEC-HPLC analysis (FIG. 7A). This result indicates that a functional MPR region (the region corresponding to residues 699-724 of SEQ ID NO: 1) is necessary for the formation of Peak II.
  • gB698-GCN4 was shown to have a higher Peak I Tm than what is usually observed for postfusion gB.
  • EBV-gB GCN4 revealed a major peak corresponding to CMV-gB GCN4 Peak II elution time (FIG. 7A), however NanoDSF revealed a Tm close to 78 °C, which is typical of postfusion gB (FIG. 7B). Further analysis can be conducted to assess and confirm the conformation of EBV-gB GCN4.
  • gB’-Q510P-GCN4 A large batch (1 L) of gB’-Q510P-GCN4 was produced to obtain enough Peak II for structural and stability studies as well as for future uses.
  • gB’-Q510P-GCN4 was initially purified by single step purification using Excel-NiNTA and followed by several runs over preparative gel filtration (used HILOAD 16/600 SUPERDEX (GE Healthcare)). The eluted proteins were tested by SEC-HPLC and NanoDSF (data not shown). SEC fractions were analyzed by NanoDSF to evaluate the relative amount of Peak I and Peak II. Fractions containing mostly Peak II were pooled together and analyzed by NanoDSF and HPLC-SEC (data not shown). The final yield was about 10 mg of 90% pure Peak II of gB’-Q510P-GCN4.
  • Peak I molecules have a conformation that corresponds to that of postfusion gB (such molecules being referred to as“post-fusion-like” herein for that reason) because its Tm ( ⁇ 78.5 °C) is similarto that of LVL759 under nanoDSF analysis and it has a more elongated shape
  • Peak II molecules have a conformation that corresponds to a non-postfusion conformation because its Tm is lower ( ⁇ 65.5 °C) and it has a more globular shape than Peak I molecules.
  • the unfolding profile of LVL759 under NanoDSC analysis suggests that Peak I molecules are also in a non-post-fusion conformation or an intermediate state between pre- and postfusion conformations.
  • FIG. 9 provides a schematic representation of what is believed to be the transition from pre- to postfusion HCMV gB based on CryoET prefusion model and postfusion crystal structure (FIG. 9 is a modified version of figure 4 from Nishimura & Mori“Chapter 8: Entry of betaherpesviruses” 2019 Ad. Vir. Res. 104: 283-312).
  • FIG. 9 is a modified version of figure 4 from Nishimura & Mori“Chapter 8: Entry of betaherpesviruses” 2019 Ad. Vir. Res. 104: 283-312).
  • the biophysical evidence collected so far suggest that Peak II should resemble to the globular state, while Peak I should be like the extended state (FIG. 9).
  • the modified HCMV gB molecule“AD169-GCN4’’ was generated by truncating the MPR (i.e., deleting the residues c-terminal to residue K723); inserting two proline residues C- terminally adjacent to residue K723 (i.e., P724 and P725 of SEQ ID NO: 14); and operably linking the c-terminal-most proline (P725) to the GCN4 with sequence SEQ ID NO: 2.
  • AD169-GCN4 can be represented by N’-K723-P-P-GCN4-C’.
  • a TEV cleavage site and a 6xHis tag (SEQ ID NO: 5) was inserted c-terminally adjacent to the GCN4. See the AD169-GCN4 amino acid sequence SEQ ID NO: 14.
  • gB’-GCN4 (based on HCMV Merlin strain, as described above) and Molecules (1)-(8) from Example 1 were expressed in 150 ml of expression media, purified by single step purification, and further analyzed (see FIG. 10A, gB’-GCN4 as“WT” therein).
  • Molecules (9)-(25) listed below (also based on HCMV Merlin strain) were expressed in 30 ml of expression media, purified by single step purification, and further analyzed (see FIG. 10B).
  • AD169-GCN4 (based on HCMV AD169 strain, as described above) and Molecules (26)-(27) were also were expressed in 30 ml of expression media, purified by single step purification, and further analyzed (see FIG. 10C, AD169-GCN4 as“WT AD169’’ therein). As is also explained elsewhere herein, residues W505 and Q509 of AD169 gB
  • each protein was analyzed by nanoDSF and showed two thermal transition related to Peak I and Peak II (data not shown).
  • SEC-HPLC was used to quantify the relative amount of Peak II and Peak I and their ratio (FIG. 10A, 10B, and 10C).
  • gB’-W506-Q510P-GCN4 produced similar amount of Peak I I as did gB’-W506P-GCN4 and gB’-Q510P-GCN4 single mutants (FIGs. 10A and 10B), indicating that it is not possible to further increase the production of Peak I I (at least in HCMV Merlin strain) above what is obtained by gB’-W506P-GCN4 and gB’-Q510P-GCN4.
  • both AD169-Q509P-GCN4 and AD169-W505P-Q509P-GCN4 both based on AD169 strain
  • Peak II an about 3-fold increase in the Peak I I to Peak I ratio compared to the ratio observed with the control AD169-GCN4 molecule (see FIG. 10C, AD169-GCN4 labeled as “WT AD169” therein)
  • WT AD169 the results in Merlin-strain-based molecules as compared to AD169-strain-based molecules is due to sequence variability, in particular, sequence variability across the furin cleavage site or at the N-terminus (which is known to be a less conserved region of gB).
  • AD169-Q509P-GCN4 The presence of several epitopes in AD169-Q509P-GCN4 was confirmed via Surface Plasmon Resonance (SPR) that showed high affinity binding to several distinct antibodies, including the presence of antigenic domain 5 (AD- 5) epitope via high affinity binding to a neutralizing antibody (antibody data not shown; see Chandramouli et al. 2015 Nat. Comm. 6(8176): 1 -12 regarding AD- 5).
  • SPR Surface Plasmon Resonance
  • introducing a R496P, W505P, C506P, D508P, D508P and Q509P, or Q509P and R510P substitution(s) (numbered according to SEQ ID NO: 6) to the AD169-GCN4 molecule is likewise expected to increase the peak I I population as compared to what was observed for AD169-GCN4.
  • a modified HCMV gB protein comprising a heterologous trimerization domain, such as a GCN4 Leucine Zipper domain (GCN4)) operably linked C-terminal to the residue corresponding to 724 numbered according to SEQ ID NO: 1 (specifically K724 numbered according to SEQ ID NO: 1 ).
  • GCN4 GCN4 Leucine Zipper domain
  • modified HCMV gB protein of 1 that does not comprise the native amino acid residues c-terminal to the residue corresponding to 724, numbered according to SEQ ID NO: 1 (specifically does not comprise the native amino acid residues corresponding to 725-907 of SEQ ID NO: 1).
  • modified HCMV gB protein of 2 wherein the native amino acid residues comprise a transmembrane domain, cytoplasmic domain, or both.
  • modified HCMV gB protein of any one of 1-3, wherein one or two proline residues are between the residue corresponding to 724, numbered according to SEQ ID NO: 1 , and the trimerization domain.
  • modified HCMV gB protein of any one of 1 -4, where the trimerization domain is a GCN4 domain comprising SEQ ID NO: 2.
  • modified HCMV gB protein of any one of 1-5 that is a modified HCMV strain AD169 gB or Merlin strain HCMV gB.
  • modified HCMV gB protein of any one of 1-6 that is a modified HCMV gB protein and the trimerization domain is operably linked c-terminal to a lysine (K) residue corresponding to 724 of SEQ ID NO: 1 (e.g., linked c-terminal to residue 724 of SEQ ID NO: 1 or 723 of SEQ ID NO: 6).
  • K lysine
  • modified HCMV gB protein of 7 that is a modified HCMV gB protein modified as compared to a HCMV gB protein from Merlin strain.
  • modified HCMV gB protein of any one of 1-8 that is in pre-fusogenic conformation.
  • the modified HCMV gB protein of 10 comprising:
  • a substitution at the fusion loop amino acids corresponding to 155, 156, 157, 240, 241 , and 242 of SEQ ID NO: 1 specifically a substitution to glycine (G) at the residue corresponding to 155 of SEQ ID NO: 1 , a substitution to histidine (H) at the residue corresponding to 156 of SEQ ID NO: 1 , a substitution to arginine (R) at the residue corresponding to 157 of SEQ ID NO: 1 , a substitution to phenylalanine (F) at the residue corresponding to 240 of SEQ ID NO: 1 , a substitution to phenylalanine (F) at the residue corresponding to 241 of SEQ ID NO: 1 , and a substitution to histidine (FI) at the residue corresponding to 242 of SEQ ID NO: 1), or
  • the modified FICMV gB protein of any one of 1-11 further comprising a purification tag (specifically a 6-histidine purification tag), optionally that is operably linked to a cleavage linker (specifically a TEV cleavage linker).
  • a purification tag specifically a 6-histidine purification tag
  • cleavage linker specifically a TEV cleavage linker
  • the modified FICMV gB protein of 13 that is:
  • a modified HCMV gB protein comprising N478P, L479P, V480P, Y481 P, A482P, Q483P, L484P, Q485P, F486P, T487P, Y488P, D489P, T490P, L491 P, R492P, G493P, Y494P, I495P, N496P, R497P, A498P, L499P, A500P, Q501 P, I502P, A503P, E504P, A505P, W506P, C507P, V508P, D509P, Q510P, R511 P, or combinations thereof, numbered according to SEQ ID NO: 1 ;
  • a modified HCMV gB protein comprising N477P, L478P, V479P, Y480P, A481 P, Q482P, L483P, Q484P, F485P, T486P, Y487P, D488P, T489P, L490P, R491 P, G492P, Y493P, I494P, N495P, R496P, A497P, L498P, A499P, Q500P, 1501 P, A502P, E503P, A504P, W505P, C506P, V507P, D508P, Q509P, R510P, or combinations thereof, numbered according to SEQ ID NO: 6 ;
  • a modified HCMV gB protein comprising N478G, L479G, V480G, Y481 G, A482G, Q483G, L484G, Q485G, F486G, T487G, Y488G, D489G, T490G, L491 G, R492G, Y494G, I495G, N496G, R497G, A498G, L499G, A500G, Q501 G, I502G, A503G, E504G, A505G, W506G, C507G, V508G, D509G, Q510G, R511 G, or combinations thereof, numbered according to SEQ ID NO: 1 ;
  • a modified HCMV gB protein comprising N477G, L478G, V479G, Y480G, A481 G, Q482G, L483G, Q484G, F485G, T486G, Y487G, D488G, T489G, L490G, R491 G, Y493G, I494G, N495G, R496G, A497G, L498G, A499G, Q500G, 1501 G, A502G, E503G, A504G, W505G, C506G, V507G, D508G, Q509G, R510G, or combinations thereof, numbered according to SEQ ID NO: 6;
  • V a modified HCMV gB protein comprising N478S, L479S, V480S, Y481 S, A482S, Q483S, L484S, Q485S, F486S, T487S, Y488S, D489S, T490S, L491 S, R492S, G493S, Y494S, I495S, N496S, R497S, A498S, L499S, A500S, Q501 S, I502S, A503S, E504S, A505S, W506S, C507S, V508S, D509S, Q510S, R511 S, or combinations thereof, numbered according to SEQ ID NO: 1 ;
  • a modified HCMV gB protein comprising N477S, L478S, V479S, Y480S, A481 S, Q482S, L483S, Q484S, F485S, T486S, Y487S, D488S, T489S, L490S, R491 S, G492S, Y493S, I494S, N495S, R496S, A497S, L498S, A499S, Q500S, 1501 S, A502S, E503S, A504S, W505S, C506S, V507S, D508S, Q509S, R510S, or combinations thereof, numbered according to SEQ ID NO: 6;
  • VII a modified HCMV gB protein comprising N478A, L479A, V480A, Y481A, Q483A, L484A, Q485A, F486A, T487A, Y488A, D489A, T490A, L491A, R492A, G493A, Y494A, I495A, N496A, R497A, L499A, Q501A, I502A, E504A, W506A, C507A, V508A, D509A, Q510A, R511A, or combinations thereof, numbered according to SEQ ID NO: 1 ; or
  • VIII a modified HCMV gB protein comprising N477A, L478A, V479A, Y480A, Q482A, L483A, Q484A, F485A, T486A, Y487A, D488A, T489A, L490A, R491A, G492A, Y493A, I494A, N495A, R496A, L498A, Q500A, 1501 A, E503A, W505A, C506A, V507A, D508A, Q509A, R510A, or combinations thereof, numbered according to SEQ ID NO: 6.
  • the modified HCMV gB protein of 14 that is:
  • a modified HCMV gB protein comprising R497P, A503P, E504P, A505P, W506P, C507P, V508P, D509P, Q510P, R511 P, or combinations thereof, numbered according to SEQ ID NO: 1 ; or
  • HCMV gB protein comprising R496P, A502P, E503P, A504P, W505P, C506P, V507P, D508P, Q509, R510P, or combinations thereof, numbered according to SEQ ID NO: 6 .
  • a modified HCMV gB protein comprising R497P, W506P, C507P, D509P, Q510P, R511 P, W506P and Q510P, D509P and Q510P, or combinations thereof, numbered according to SEQ ID NO: 1 ; or
  • a modified HCMV gB protein comprising R496P, W505P, C506P, D508P, Q509P, R510P, W505P and Q509P, D508P and Q509P, or combinations thereof, numbered according to SEQ ID NO: 6 (such as a modified HCMV gB protein comprising Q509P, W505P and Q509P, or combinations thereof, numbered according to SEQ ID NO: 6).
  • modified HCMV gB protein of any one of 1-16 that is a modified HCMV gB protein comprising the substitution Q510P numbered according to SEQ ID NO: 1.
  • the modified HCMV gB protein of any one of 1-18 comprising an amino acid sequence with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3 or 4 (such as a modified HCMV gB protein with an amino acid sequence comprising: residues 23-759 of SEQ ID NO: 3, residues 23-733 of SEQ ID NO: 11 , residues 22-740 of SEQ ID NO: 13, residues 23-758 of SEQ ID NO: 14, or one or SEQ ID NOs: 15-30).
  • the modified HCMV gB protein of 21 wherein the nanoparticle is, or is derived from, a lumazine synthase or ferritin protein.
  • An isolated nucleic acid comprising a polynucleotide sequence encoding the modified HCMV gB protein of any one of 1-19.
  • RNA is a self-replicating RNA, optionally an alphavirus replicon.
  • VRP alphavirus replication particle
  • An immunogenic composition comprising the modified HCMV gB protein of any one of 1- 22, the nucleic acid of any one of 23-26, or the VRP of 27.
  • the immunogenic composition of 28 further comprising an adjuvant.
  • a recombinant vector comprising the nucleic acid of any one of 23-26.
  • An isolated host cell comprising the nucleic acid of any one of 23-26, optionally wherein the polynucleotide is DNA and stably incorporated into the genomic DNA of the host cell.
  • the isolated host cell of 33, wherein the mammalian cell is a CHO cell or HEK-293 cell.
  • a cell culture comprising the host cell of any one of 32-34.
  • a process of producing a modified HCMV gB protein comprising culturing the host cell of any one of 32-34 under suitable conditions, thereby expressing the modified HCMV gB protein.
  • a modified HCMV gB protein produced by the process of 36 or 37.
  • a process of producing a population of host cells comprising culturing host cells according to any one of 32-34 under suitable conditions for expression of a modified HCMV gB protein according to any one of 13-20, thereby producing a population of host cells and optionally purifying the modified HCMV gB protein, wherein at least 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, or 45% of the purified modified HCMV gB proteins are in pre-fusogenic conformation.
  • An immunogenic composition comprising the modified HCMV gB protein of any one of 1- 22 or 38 and at least one additional HCMV protein, or an immunogenic fragment thereof.
  • the immunogenic composition of 42, wherein the at least one additional HCMV protein, or immunogenic fragment thereof, is gO, gH, gl_, pUL128, pUL130, pUL131 , pp65, IE1 , or a combination thereof.
  • the immunogenic composition of 42 or 43 comprising the modified HCMV gB protein of any one of 1-22 and 38; gH, or an immunogenic pentamer-complex forming fragment thereof; gl_, or an immunogenic pentamer-complex forming fragment thereof; pUL128, or an immunogenic pentamer-complex forming fragment thereof; pUL130, or an immunogenic pentamer-complex forming fragment thereof; and pUL131 , or an immunogenic pentamer-complex forming fragment thereof.
  • the immunogenic composition of 47 wherein the adjuvant comprises aluminum salt, a TLR7 agonist, TLR4 agonist, MPL, 3D-MPL, saponin, or an oil-in-water emulsion.
  • a pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of 49.
  • VRP of 27 immunogenic composition of any one of 28-30, 42-48; vector of 31 ; antibody or antibody fragment of 49; or pharmaceutical composition of 50 in the prevention or treatment of a HCMV infection.
  • VRP of 27 immunogenic composition of any one of 28-30, 42-48; vector of 31 ; antibody or antibody fragment of 49; or pharmaceutical composition of 50 for inducing an immune response against HCMV.
  • modified HCMV gB protein of any one of 1-22 or 38; nucleic acid of any one of 24-26; VRP of 27; immunogenic composition of any one of 28-30, 42-48; vector of 31 ; antibody or antibody fragment of 49; or pharmaceutical composition of 50 for the manufacture of a medicament for inhibiting HCMV entry into a cell (specifically, inhibiting membrane fusion for HCMV entry into a cell).
  • VRP of 27 immunogenic composition of any one of 28-30, 42-48; vector of 31 ; antibody or antibody fragment of 49; or pharmaceutical composition of 50 for use in inhibiting HCMV entry into a cell (specifically, inhibiting membrane fusion for HCMV entry into a cell).
  • a method of inhibiting HCMV entry into a cell comprising contacting the cell with the modified HCMV gB protein of any one of 1-22 or 38; nucleic acid of any one of 24-26; VRP of 27; immunogenic composition of any one of 28-30, 42-48; vector of 31 ; antibody or antibody fragment of 49; or pharmaceutical composition of 50.
  • a method of inducing an immune response against HCMV in a subject comprising administering to the subject an immunologically effective amount of the modified HCMV gB protein of any one of 1 -22 or 38; nucleic acid of any one of 24-26; VRP of 27; immunogenic composition of any one of 28-30, 42-48; vector of 31 ; antibody or antibody fragment of 49; or pharmaceutical composition of 50.
  • kits for identifying the presence of a modified HCMV gB protein within a composition for the purification of a modified HCMV gB protein from a composition, for detecting a modified
  • HCMV gB protein in pre-fusion, or for detecting a modified HCMV gB protein in a non-post-fusion conformation, or for the prevention or treatment of HCMV infection; comprising the antibody or antigen-binding fragment thereof of 48, and optionally instructions for using the antibody or antigen-binding fragment thereof.
  • SEQ ID NO: 1 Amino acid sequence of wild type, Merlin strain, Human Cytomegalovirus gB protein. See Chandramouli et al. 2015 Nat. Comm. 6(8176) at figure 1 : See also UniProtKB Accession No. F5HB53 (GB_HCMVM; sequence version 1 last updated June 28, 2011).
  • SEQ ID NO: 3 (gB’-GCN4) - exemplary amino acid sequence of a modified HCMV gB protein from these studies comprising, as compared to wild type Merlin HCMV gB (SEQ ID NO: 1):
  • This sequence may be further modified by operably linking a TEV cleavage site and
  • 6xHis tag (SEQ ID NO: 5) to the C-terminus of the GCN4 sequence, as is shown in SEQ ID NO: 4.
  • SEQ ID NO: 5 i.e., a TEV cleavage site (underlined) and 6xHis tag (double underlined) C-terminally adjacent to the GCN4 sequence.
  • SEQ ID NO: 5 - a TEV cleavage site (underlined) and 6xHis tag (double underlined).
  • SEQ ID NO: 6 AD169 Strain HCMV gB amino acid sequence corresponding to UniProtKB Accession P06473 (version 1 of sequence last updated January 1 , 1988; see also figure 2 of0 Burke & Heldwein 2015 PLOS Path. DOI: 10.1371).
  • SEQ ID NO: 11 (gB698-GCN4 with TEV and 6xHis (SEQ ID NO: 5)) - exemplary amino acid sequence of a modified HCMV gB protein from these studies comprising, as compared to wild type Merlin HCMV gB (SEQ ID NO: 1):
  • SEQ ID NO: 5 i.e., a TEV cleavage site (underlined) and 6xHis tag (double underlined) C-terminally adjacent to the GCN4 sequence.
  • SEQ ID NO: 12 the wild type EBV gB sequence published as UniProtKB Accession P03188 (entry 1 10 dated December 11 , 2019) and that is characterized by having the following features:
  • Cytoplasmic domain is from residues R754-F857 (underlined).
  • SEQ ID NO: 13 (EBV-GCN4) - exemplary amino acid sequence of a modified EBV gB protein from these studies comprising, as compared to EBV gB sequence SEQ ID NO: 12 above:
  • SEQ ID NO: 5 i.e., a TEV cleavage site (underlined) and 6xFlis tag (double underlined) C-terminally adjacent to the GCN4 sequence.
  • SKNYHLENEV ARLKKLVGER ENLYFOGGHH HHHH SEQ ID NO: 14 (AD169-GCN4) - exemplary amino acid sequence of a modified HCMV AD169 strain gB protein from these studies comprising, as compared to HCMV AD169 gB sequence SEQ ID NO: 6 above:
  • SEQ ID NO: 5 i.e., a TEV cleavage site (underlined) and 6xHis tag (double underlined) C-terminally adjacent to the GCN4 sequence.
  • SEQ ID NO: 15 (gB’-R497P-GCN4) - residues 23-759 of SEQ ID NO: 3 further modified by the R497P substitution (is R475P numbered according to this sequence).
  • SEQ ID NO: 16 (gB’-W505P-GCN4) - residues 23-759 of SEQ ID NO: 3 further modified by the W506P substitution (is W484P numbered according to this sequence).
  • SEQ ID NO: 20 (gB’-R511 P-GCN4) - residues 23-759 of SEQ ID NO: 3 further modified by the R511 P substitution (is R489P numbered according to this sequence).
  • SEQ ID NO: 21 (gB’-W506P-Q510P-GCN4) - residues 23-759 of SEQ ID NO: 3 further modified by the two W506P and Q510P substitutions (are W484P and Q488Pnumbered according to this sequence).
  • SEQ ID NO: 22 (gB’-D509P-Q510P-GCN4) - residues 23-759 of SEQ ID NO: 3 further modified by the two D509P and Q510P substitutions (are D487P and Q488P numbered according to this sequence).
  • SEQ ID NO: 24 (AD169-W505P-GCN4) - residues 23-758 of SEQ ID NO: 14 further modified by the W505P substitution (is W483P numbered according to this sequence).
  • SEQ ID NO: 25 (AD169-C506P-GCN4) - residues 23-758 of SEQ ID NO: 14 further modified by the C506P substitution (is C484P numbered according to this sequence).
  • SEQ ID NO: 26 (AD169-D508P-GCN4) - residues 23-758 of SEQ ID NO: 14 further modified by the D508P substitution (is D486P numbered according to this sequence).
  • SEQ ID NO: 28 (AD169-R510P-GCN4) - residues 23-758 of SEQ ID NO: 14 further modified by the R510P substitution (is R488P numbered according to this sequence).
  • SEQ ID NO: 29 (AD169-W505P-Q509P-GCN4) - residues 23-758 of SEQ ID NO: 14 further modified by the two W505P and Q509P substitutions (are W483P and Q487P numbered according to this sequence).
  • SEQ ID NO: 30 (AD169-D508P-Q509P-GCN4) - residues 23-758 of SEQ ID NO: 14 further modified by the two D508P and Q509P substitutions (are D486P and Q487P numbered according to this sequence).

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